Austin Community College
Biotechnology Department
Standard Operating Procedures
9th Edition, Fall 2012
Table of Contents
Page
ACC-001
AUT-001
AUT-003
Bio-Rad TC10 Automated Cell Counter (REV A)
VWR/Thermo Scientific Sterilemax Autoclave (REV C)
Sterilmatic Autoclave (REV A)
3
5
8
BAL-001
BAL-002
BAL-003
BAL-004
BAL-005
BSC-001
Acculab V1-200 balance (REV A)
Mettler-Toledo Excellence XS analytical balance (REV A)
Ohaus AP110 analytical balance (REV A)
Mettler-Toledo PL1502-S balance (REV A)
Ohaus Scout Pro balance (REV C)
Nu-Aire NU-427 Biosafety Cabinet (REV A)
10
12
14
16
18
20
CEN-001
CEN-002
CEN-003
CEN-004
CUV-001
Eppendorf 5417R Refrigerated Microcentrifuge (REV A)
Eppendorf 5804R Refrigerated Microcentrifuge (REV A)
VWR Clinical 200 Centrifuge (REV A)
VWR Galaxy 16D Microcentrifuge (REV A)
Selection, Use, and Cleaning of Cuvettes (REV A)
22
23
25
26
27
DTR-001
Digital Temperature Recorder (REV A)
30
ELC-002
ELC-003
ELC-004
EPR-001
Bio-Rad Mini-Protean Vertical Electrophoresis System (REV A)
Bio-Rad Mini Trans-blot Electrophoretic Transfer System (REV A)
Edvotek Electrophoresis Chambers (M12 and Hexagel) (REV A)
Eppendorf Repeater Plus Pipettor (REV A)
31
35
38
41
FLU-002
FPLC-001
Varian Cary Fluorometer (REV A)
DuoFlow Forward Phase Liquid Chromatography System (REV B)
43
45
GDS-003
GDS-004
GWC-001
ChemiDoc Imaging System with Quantity One Software (REV A)
ChemiDoc Imaging System with Image Lab Software (REV B)
Cleaning Lab Glassware (REV B)
52
55
58
HPLC-001
HPLC-002
Waters Breeze HPLC System (REV A)
Dionex Ultimate HPLC System with Manual Inject Valve (REV A)
59
62
INC-001
HERAcell CO2 incubator (REV A)
69
MCP-001
MIC-001
MIC-002
MPR-002
VWR micropipettors (REV A)
Nikon TS100 Inverted Microscope (REV A)
Nikon Eclipse 55i Fluorescence Microscope (REV A)
BioTek Synergy Multimode Plate Reader (REV A)
72
77
80
85
PCR-001
PCR-004
PCR-005
Bio-Rad MyIQ Real-time qPCR System (REV A)
Applied Bio 2720 Thermal Cycler (REV A)
Bio-Rad CFX96 Real-time qPCR System (REV B)
88
94
96
2
PHM-002
Accumet AB15 pH meter (REV A)
102
RCV-001
Receiving and Storing Materials (REV A)
105
SOL-001
SPC-001
SPC-002
SPC-003
SPC-004
SPC-008
Labeling solutions (REV A)
Genesys 10 Spectrophotometer (REV A)
BioMate 3 Spectrophotometer (REV A)
Nanodrop 1000 Spectrophotometer (REV A)
Perkin-Elmer Lambda Bio+ Spectrophotometer (REV A)
Nanodrop 2000 Spectrophotometer (REV A)
107
110
114
118
119
122
TAC-001
Tachometer (REV A)
124
VAC-001
Integra VacuSafe Aspirator (REV A)
126
WPS-001
RODI Water Purification System (REV A)
127
Record of Revisions to Standard Operating Procedures
129
3
SOP ACC-001
TC10 AUTOMATED CELL COUNTER
Revision A, June 7, 2011
Title: Bio-Rad TC10 Automated Cell Counter
1.0 SCOPE AND APPLICATION:
This SOP outlines the procedure for use and maintenance of the Bio-Rad TC10 Automated Cell
Counter.
2.0 SUMMARY OF METHOD
MATERIALS
1.
2.
3.
4.
5.
TC10 Automated Cell Counter
TC10 Sample slide
Trypan blue stain
Cell sample
P20 micropipettor and tips
PART A: BASIC ABSORBANCE, TRANSMITTANCE,
AND CONCENTRATION READINGS
AVOID touching the optical surface of the
slides.
1. Turn on the TC10 Automated Cell Counter via
the green square button on side bottom of
equipment.
2. Wait for the instrument to initialize.
3. Prepare slide to be analyzed while waiting.
4. Select options on the home screen by pressing
the [UP] or [DOWN] key on the navigation key
pad.
5. When your selection is highlighted in green,
press [ENTER] on the navigation key pad.
6. Choose Count Cells and insert the prepared
sample slide into the slide port of the cell
counter. The counter will focus and count cells.
7. The Current Count screen will appear and
display the total count of your sample, the
date and time, and the option to calculate the
dilution of your sample. In this screen, you will
also be able to view an image of your sample,
print data, and view a histogram. To toggle
between these options press the [UP] or
[DOWN] key and press [ENTER].
Loading slide using trypan blue stain
Combine 10 µl cell suspension with 10 µl of
trypan blue dye in a microcentrifuge tube.
Pipet gently up and down to mix.
Pipet 10 µl of the mixture into a TC10
sample slide. Place pipet tip at 45 degree
angle at the bottom of sample loading
area (the half-circle at outer end of the
chamber). Only load into the outer end of
the counting slide. The center notches are
for distribution purposes.
4
8. Any time you want to return to the Home
screen press the [HOME] key on the navigation
key pad.
The cell counter demonstrates high
reproducibility within the range of 5X104 to
1X107 cells/mL.
9. The TC10 Automated cell counter allows you to
print if connected to a printer. Select “Previous
Counts” using the [UP] or [DOWN] key press
[ENTER] and press [ENTER] again to print.
Throw away the slide after they are used.
These slides may not be reused.
10. You may save counts and images to a USB
storage device. Insert a device into the USB
port. Select Export Previous Counts on the
Home screen and press [ENTER] to send to the
device.
11. Use the Dilution Calculator to find the
concentration of your sample.
5
SOP AUT-001
OPERATION OF VWR/THERMO SCIENTIFIC STERILEMAX AUTOCLAVE
Revision C, August 10, 2012
Title: Operation of VWR/Thermo Scientific Sterilemax Autoclave
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for operation of the Sterilemax autoclave.
2.0 SUMMARY OF METHOD
MATERIALS
COMMENTS
1. Sterilemax autoclave
2. 4-7 L deionized water
3. cleaning solution and scouring pad
PART A: OPERATION
1. Plug autoclave into a 115-V outlet. Turn
on autoclave using switch on bottom
right edge of front of machine.
2. Check water level by opening reservoir
on top right side. Water should be at
least six inches from bottom of reservoir
and should not be discolored or
cloudy.
This autoclave automatically cycles water
back into the reservoir, so the water is often
contaminated. Water should be drained
and replaced at least once a week.
3. If water appears contaminated, empty
the reservoir by following instructions in
Part B. If water level is too low, add tap
water until water is at least 6 inches
high. Replace lid on reservoir.
4. Open chamber door by turning handle
counter-clockwise until handle swings
outward. Squeeze the door release
panel on bottom left edge of door to
release the door fully. Pull forward to
open.
5. Load items to be autoclaved into
chamber. There are three racks which
are all removable, but never operate
the autoclave without the lowest rack
or glassware could break.
6. Close the chamber door and swing the
handle to the right until its axis is snugly
This autoclave will accommodate three 2-L
flasks, but nothing taller than that.
Be sure to mark items with a small piece of
autoclave tape and check for darkened
stripes to indicate sterilization.
6
placed in the door. Turn the handle
clockwise until it is very difficult to turn
further.
The packs cycle is designed for lab packs,
which are containers full of biohazardous
sharps and Plaster of Paris. You will not
need to use this cycle.
7. Press “start” to choose a program.
Each program is suited to sterilize a
particular material.
8. If you are sterilizing liquids, choose the
liquid cycle. This will heat to a higher
temperature than the other cycles and
exhaust more slowly to prevent
evaporation and splashing. Be certain
that all caps have been loosened or
containers can explode under high
pressure!
9. For wrapped materials (such as
sterilizing pouches full of instruments or
for biohazard-contaminated materials
in a biohazard bag), choose the
wrapped cycle. This will exhaust quickly.
10. For unwrapped materials (such as a
fermentation vessel or other empty
glassware), choose the unwrapped
cycle. This will exhaust very quickly.
11. Once you have pressed the button
corresponding to the program you
want, the parameters of the program
will appear. You can change these if
you wish, but in most cases you will
want to leave the default parameters.
12. Press “start” and the program will
begin.
13. When the sterilization is complete, the
machine will beep and display a
command to open the chamber door
to allow the chamber to exhaust.
14. Allow the drying cycle to complete
before you attempt to remove items,
especially hot liquids.
15. When drying is complete, open door
completely and stay to the right to
avoid steam burns.
16. Remove trays or items using insulated
Open chamber as described in step 4, but
stay to the right side and keep your hand
and arm away from the opening so you
are not burned by steam escaping.
7
gloves or hot hands to avoid burns.
Place on a lab bench or cart to cool to
a reasonable temperature before
attempting to use.
PART B: MAINTENANCE
1. At least once weekly, the reservoir
should be drained completely of water
and refilled. To drain, open the
chamber door when cool. Locate the
drain hose and find the working end
(has a white plastic gasket).
2. Place the non-working end into the
sink. Insert the working end of the drain
hose into the drain plug, located inside
the door frame on the lower right.
3. Reservoir will begin draining
immediately. Drain completely and
remove hose by pressing on release
button (just below the drain plug).
4. Refill reservoir with 4-7 liters of deionized
water.
5. At least once monthly, the autoclave
chamber should be scrubbed using a
plastic scouring pad and a special
cleaner (found on top of the machine).
PART C: CHECKLIST
□
Check for adequate water
□
Loosen caps on bottles and DO
NOT OVERFILL (more than 1/3 full)
□
Load chamber, close and start
cycle
□
Remove your items when finished
8
SOP AUT-003
USE AND MAINTENANCE OF STERILMATIC AUTOCLAVE
Revision A, February 22, 2011
Title: Use and Maintenance of Sterilmatic Autoclave
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for using the Sterilmatic autoclave.
2.0 SUMMARY OF METHOD
MATERIALS
COMMENTS
1. Sterilmatic autoclave
2. 4 L tap water
PART A: OPERATION
1. Load items into chamber. Do not
crowd the items too closely, or the
steam will not penetrate between
them.
2. Make sure chamber drain is in closed
position. Pour 4 L of tap water into the
chamber until it reaches the fill line. Do
not exceed the fill line!
3. Grasp the door handle and hold it in a
vertical position while pulling down until
bottom of door rests in the door
opening. Make sure the rubber seal is
flush with the door. Then push door
handle down to engage the lock.
4. Select the correct exhaust type.
“Liquids” is to be used for any liquids,
while “Instruments” can be used for all
other items.
5. Set the temperature to 121ºC.
6. If you are sterilizing liquids, choose the
liquid cycle. This will heat to a higher
temperature than the other cycles and
exhaust more slowly to prevent
evaporation and splashing. Be certain
that all caps have been loosened or
containers can explode under high
pressure!
Mark items with indicator tape. Make sure
that closed vessels, especially glass bottles,
are vented (lid not entirely closed).
9
7. Turn the timer to start operation. Timer
should always be set to at least 15
minutes, as this is the minimum time for
sterilization to be effective.
8. When the chamber reaches the
selected temperature, the timer will
begin.
9. When cycle is complete, allow the
chamber pressure to drop to zero
before attempting to open the door.
10. Open door completely by pulling up on
the door handle. Stay to the side to
avoid steam burns.
11. Remove trays or items using insulated
gloves or hot hands to avoid burns.
Place on a lab bench or cart to cool to
a reasonable temperature before
attempting to use.
PART B: MAINTENANCE
At the end of each day, the chamber
should be drained completely. Place a
bucket under the drain and open drain
valve. Close valve when draining is
complete and dispose of water (do not reuse).
PART C: CHECKLIST
□ Close drain valve and add 4 L tap
water to the chamber
□
Loosen caps on bottles and DO
NOT OVERFILL (more than 1/3 full)
□
Load chamber and close door
□
Choose appropriate cycle
□
Start timer
□
Remove your items when finished
□
Open the drain valve to drain the
chamber
10
SOP BAL-001
OPERATION AND CALIBRATION OF ACCULAB MODEL VI-200 ELECTRONIC BALANCE
Revision A, February 22, 2011
Title: Operation and calibration of Acculab V1-200 Balance
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for the proper operation and calibration of the
Acculab Model VI-200 Balance.
2.0 SUMMARY OF METHOD
MATERIALS
COMMENTS
1. VI-200 balance
2. standard 200 g mass
PROCEDURE
PART A: CALIBRATION
1. Turn on balance and allow it to warm
up for at least 30 minutes.
Use only a 12V AC adapter with a negative
tip to supply power. Unplug when not in
use.
2. Remove all items from the weighing
pan and tare the balance by pressing
the TARE key.
3. Check that the balance is in gram
mode, indicated by a stable arrow on
the display next to the g symbol on the
casing.
If the balance is not in gram mode, press
and release the CAL/MODE key until the
arrow is next to g.
4. After observing a stable zero reading
(0.00) press and hold the CAL/MODE
key. After four seconds the unit will
beep and the calibration weight (+200)
will appear on the display. Release the
CAL/MODE key.
5. Gently place the 200 gram standard
mass on the weighing pan.
6. The + sign will disappear from the
display. Wait seven to ten seconds and
the unit will beep.
7. The displayed weight (200) will
disappear and then reappear as an
active value. Calibration is now
complete.
Do not disturb the balance during
calibration, and avoid vibrations and air
currents.
If the calibration weight value remains on
the display, an improper weight has been
used. Select the proper weight (200g) and
repeat the calibration.
11
PART B: OPERATION
1. Remove all items from the weighing
pan and tare the balance by pressing
the TARE key.
2. Check that the balance is in gram
mode, indicated by a stable arrow on
the display next to the g symbol on the
casing.
3. Place a weigh boat, weigh paper, or
small vessel (such as an Erlenmeyer
flask) on the balance pan.
4. Press the TARE key to tare the weight of
the weighing vessel.
5. Place the substance to be weighed
into the weighing vessel and allow the
value to stabilize on the display.
6. Record the mass of the substance,
remove the weighing vessel and
contents from the balance and turn off.
7. Check that the entire balance and the
area around it are clean and dry.
Reagents can be weighed directly into any
vessel as long as it does not weigh more
than 200 grams (the maximum capacity of
this model).
If weighing a chemical, use a clean, dry
spatula to transfer it into the vessel, or if a
free-flowing solid, gently sift it from the
container.
Record any error messages or drifting mass
values in the comments section of the log.
If the balance was dirty or wet or the
balance area was messy when you began
working, record that in the comments
section.
12
SOP BAL-002
OPERATION AND CALIBRATION OF
METTLER-TOLEDO EXCELLENCE XS MODEL ANALYTICAL BALANCE
Revision A, May 14, 2010
Title: Operation and calibration of M-T Excellence analytical balance
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for the proper operation, calibration and
maintenance of the M-T Excellence analytical balance.
2.0 SUMMARY OF METHOD
MATERIALS
1. VI-200 balance
2. standard 200 g mass
PROCEDURE
PART A: CALIBRATION
The Excellence XS analytical balance has
fully automatic calibration functions, so it
needs to be calibrated by hand very
rarely.
COMMENTS
Use of this SOP indicates that the user has
read the M-T Excellence XS user manual.
The manual must be read prior to use of
this SOP.
PART B: OPERATION
1. CAUTION: Do not overload balance.
Maximum capacity is 220 g.
2. Place a weigh boat or paper on the
weighing pan, close all draft shields,
and tare the balance by pressing the
TARE key. “Net” will appear in the
display to indicate that all displayed
weights will be net values.
3. Open the draft shield and insert the
items to be weighed. Never sift
chemicals from a container directly
into the analytical balance. Spillage
could devastate the instrument.
Instead, carefully transfer material using
a spatula.
4. Close the draft shield and wait for the
stability detector icon, a small circle,
disappears from the display. The
presence of this icon indicates that the
balance is NOT stable and you should
wait for it to disappear.
Do not use the ZERO key for taring. This is
only used for initial setup of the balance.
Do not disturb the balance during
operation, and avoid vibrations and air
currents.
If weighing a chemical, use a clean, dry
spatula to transfer it into the vessel.
Record any error messages or drifting mass
values in the comments section of the
balance log, found in the equipment log
book. If the balance was dirty or wet or the
balance area was messy when you began
working, record that in the comments
section.
13
5. Record the mass and remove all items
from the weighing pan. Turn off
balance by pressing the ON/OFF key
and holding down until balance is off.
6. Check that the entire balance and the
area around it are clean and dry.
Remove any material from the inside of
the balance by gently sweeping it out
with a brush. Avoid pressing down on
the balance pan while cleaning. Do
not use water to clean. Wipe any
material away from the area around
the balance. Clean spatulas or other
weighing tools thoroughly with soapy
water and dry completely before
replacing.
Never replace a used spatula to its storage
container before cleaning it thoroughly.
14
SOP BAL-003
OPERATION AND CALIBRATION OF
OHAUS AP110 ANALYTICAL BALANCE
Revision A, September 11, 2007
Title: Operation and calibration of Ohaus AP110 analytical balance
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for the proper operation, calibration and
maintenance of the Ohaus analytical balance.
2.0 SUMMARY OF METHOD
MATERIALS
1. Ohaus AP110 balance
2. standard 100 g mass
PROCEDURE
PART A: CALIBRATION
1. Make sure there is no load on the
balance pan and close the draft shield.
2. Hold down the ON/TARE button until
“MENU” appears.
3. Press the ON/TARE button once and
“AUTO” will appear.
4. Press the MODE button twice until
“User” appears.
5. Press the ON/TARE button and
“100.0000” should appear. This is the
value used to last calibrate the
balance. Press ON/TARE repeatedly to
confirm each digit of this number is
correct, then press again and “0 g”
should appear.
6. Press ON/TARE once more to start zero
mass calibration. “-C-“ should appear
and the balance will initiate calibration.
7. When “CAL 100g” appears, place a
100-g mass on the balance pan. Press
ON/TARE to initiate 100 g calibration.
8. “200.0000” should appear. Calibration
is complete.
COMMENTS
Use of this SOP indicates that the user has
read the Ohaus AP110 user manual. The
manual must be read prior to use of this
SOP.
15
PART B: OPERATION
CAUTION: Do not overload balance.
Maximum capacity is 100 g.
1. Turn balance on by pressing the
ON/TARE button.
2. Place a weigh boat or paper on the
weighing pan, close all draft shields,
and tare the balance by pressing the
TARE key.
3. Open the draft shield and insert the
items to be weighed. Never sift
chemicals from a container directly
into the analytical balance. Spillage
could devastate the instrument.
Instead, carefully transfer material using
a spatula.
4. Close the draft shield and wait for the
stability indicator, a small “s”, to
appear on the left of the display.
If weighing a chemical, use a clean, dry
spatula to transfer it into the vessel.
Record any error messages or drifting mass
values in the comments section of the
balance log, found in the equipment log
book. If the balance was dirty or wet or the
balance area was messy when you began
working, record that in the comments
section.
5. Record the mass and remove all items
from the weighing pan.
6. Turn off balance by pressing the
ON/OFF key and holding down until
balance is off.
7. Check that the entire balance and the
area around it are clean and dry.
Remove any material from the inside of
the balance by gently sweeping it out
with a brush. Wipe any material away
from the area around the balance.
Avoid pressing down on the balance pan
while cleaning. Do not use water to clean.
8. Clean spatulas or other weighing tools
thoroughly with soapy water and dry
completely before replacing.
Never replace a used spatula to its storage
container before cleaning it thoroughly.
16
SOP BAL-004
OPERATION AND CALIBRATION OF METTLER-TOLEDO PL1502-S ELECTRONIC BALANCE
Revision A, February 22, 2011
Title: Operation and calibration of Mettler-Toledo PL1502-S Balance
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for the proper operation and calibration of the
Mettler-Toledo PL1502-S Balance.
2.0 SUMMARY OF METHOD
MATERIALS
COMMENTS
1. PL1502-S balance
2. standard 200 g mass
PROCEDURE
PART A: CALIBRATION
1. Turn on balance and allow it to warm
up for at least 30 minutes.
Do not disturb the balance during
calibration, and avoid vibrations and air
currents.
2. Remove all items from the weighing
pan and tare the balance by pressing
the TARE key.
3. Press the CAL key to start calibration.
4. The maximum weight value, 200.000
grams, will flash on the LCD.
5. Gently place the 200 gram standard
mass on the weighing pan.
6. When 0.00 g flashes on the LCD,
remove the 200 g weight.
7. The adjusting is finished when the
message CAL done appears on the
display, followed by 0.00 g.
PART B: OPERATION
1. Place a weigh boat, weigh paper, or
small vessel (such as an Erlenmeyer
flask) on the balance pan.
2. Press the 0/T key to tare the weight of
Reagents can be weighed directly into any
vessel as long as it does not weigh more
than 200 grams (the maximum capacity of
this model).
If weighing a chemical, use a clean, dry
17
the weighing vessel.
3. Place the substance to be weighed
into the weighing vessel and allow the
value to stabilize on the display. The
stability detector “º” will disappear
when the reading is stable.
4. Record the mass of the substance,
remove the weighing vessel and
contents from the balance and turn off.
5. Check that the entire balance and the
area around it are clean and dry.
spatula to transfer it into the vessel, or if a
free-flowing solid, gently sift it from the
container.
18
SOP BAL-005
OPERATION AND CALIBRATION OF OHAUS SCOUT PRO ELECTRONIC BALANCE
Revision C, June 6, 2012
Title: Operation and calibration of Ohaus Scout Pro SP402 Balance
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for the proper operation and calibration of the
Ohaus Scout Pro Balance.
2.0 SUMMARY OF METHOD
MATERIALS
COMMENTS
1. Scout Pro balance
2. standard 200 g mass
PROCEDURE
PART A: CALIBRATION
1. Remove all items from the weighing
pan.
2.
Do not disturb the balance during
calibration, and avoid vibrations and air
currents.
Press and hold the ON/ZERO key until
“MENU” appears on the display.
3. After you release the ON/ZERO key,
“CAL” will display on the screen.
4. Press the ON/ZERO key again to start
calibration.
5. “C” will appear on the display and the
balance will calibrate to mass = 0 g. Do
not place anything on the balance at
this time.
6. Press the ON/ZERO key to display “C
200” (flashing).
7. Gently place the 200 gram standard
mass on the weighing pan.
8. Press the ON/ZERO key to calibrate with
the 200 gram mass. “C” will flash on the
screen for a moment, then the screen
will display “done” briefly before going
into live measurement mode.
9.
Remove the 200 gram mass.
The 0 gram calibration step happens
automatically and very quickly, so it may
appear that the balance is skipping over it.
19
PART B: OPERATION
1. With the balance on, place a weigh
boat, weigh paper, or small vessel
(such as an Erlenmeyer flask) on the
balance pan.
2. Press the ON/ZERO key to tare the
weight of the weighing vessel.
3. Place the substance to be weighed
into the weighing vessel and allow the
value to stabilize on the display. The
stability detector “*” will disappear
when the reading is stable.
4. Record the mass of the substance,
remove the weighing vessel and
contents from the balance and turn off
by pressing and holding the ON/ZERO
key.
5. Check that the entire balance and the
area around it are clean and dry.
Reagents can be weighed directly into any
vessel as long as it does not weigh more
than 200 grams (the maximum capacity of
this model).
If weighing a chemical, use a clean, dry
spatula to transfer it into the vessel, or if a
free-flowing solid, gently sift it from the
container.
20
SOP BSC-001
USE AND MAINTENANCE OF NU-AIRE BIOSAFETY CABINET
Revision A, January 25, 2011
Title: Use and Maintenance of Nu-Aire NU-427 Biosafety Cabinet
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for use of the Nu-Aire NU-427 Biosafety Cabinet.
2.0 SUMMARY OF METHOD
MATERIALS
1. Nu-Aire NU-427 Biosafety Cabinet
2. 70% alcohol, paper towels
PART A: OPERATION
1. This biosafety cabinet (“BSC”) is
operated via a touch screen. To start
up, firmly touch the screen in the top
left corner. You will hear a beep and
the BSC will start up after a few
moments.
2. Touch the blower icon in the top left
corner of the touch screen. The blower
will come on and the icon will turn
green.
3. Allow the cabinet to warm up for 15
minutes.
4. Touch the third icon from the left to turn
on the fluorescent cabinet lights.
5. When the cabinet is ready to use, raise
the window no higher than the
maximum height (marked on the side
of the cabinet) and clean the cabinet
with alcohol.
6. Begin your work by wiping down your
gloved hands and all materials to be
placed in the hood, with 70% alcohol.
7. While working in the hood, avoid
blocking the laminar flow by not
placing any objects on top of the front
grille. Keep a minimum of objects in
the hood at all, to prevent blocking the
COMMENTS
21
air flow.
8. As you work in the hood, keep sterile
materials further back than nonsterile
materials and avoid reaching over
sterile containers, especially open
containers of media. Refrain from
excess talking and do not remove your
hands from the cabinet until your work
is finished.
9. When you are finished working in the
hood, clean up materials (especially
spilled nutrient medium that might grow
contaminants) and wipe the work
surface down with 70% alcohol.
10. Touch the blower icon to turn off the
blower. Touch the UV light icon to turn
on the UV light. This is on a timer so it will
turn itself off after 15 minutes. The
window must be completely closed for
the light to turn on.
PART B: MAINTENANCE
1. Every 12 months the hoods must be
certified by a licensed technician. The
technician will also replace HEPA filters
and do any other preventive
maintenance as needed.
PART C: TROUBLESHOOTING
If the blower icon is red or blue, there is a
problem with the blower. An error message
should appear on the screen.
“Low inflow limit” means that there is a
problem with the exhaust blower. Contact
ACC Maintenance staff to check the
blowers.
22
SOP CEN-001
USE OF EPPENDORF 5417R MICROCENTRIFUGE
Revision A, April 22, 2011
Title: Use and Maintenance of Eppendorf 5417R Microcentrifuge
1.0 SCOPE AND APPLICATION:
This SOP outlines the use and maintenance of the Eppendorf 5417R refrigerated microcentrifuge.
2.0 SUMMARY OF METHOD
PROCEDURE
COMMENTS
1. Turn centrifuge on with main power
switch.
2. Press the LID button to open the
centrifuge lid.
3. Open the rotor cover by rotating the
center knob and pulling up on the
cover.
4. Load tubes into centrifuge in a
balanced configuration.
This will reduce the risk that the centrifuge
becomes unbalanced and vibrating.
Additionally, samples may be thrown out if
unbalanced, causing extreme damage.
5. Replace rotor cover. Press down on
center knob and rotate to lock in
place.
6. Close centrifuge lid and press down to
lock in place.
7. Set temperature, time, and speed
values to the appropriate settings using
the cursor keys. To change speed units
from “rpm” to “rcf”, press both cursor
keys simultaneously.
8. Press the START key to begin the run.
9. If needed, press the STOP key to stop
the run before the elapsed time. Allow
the rotor to slowly decrease speed.
10. When the centrifuge can be safely
opened, the OPEN key green LED will
light up.
11. Press OPEN to open the lid and remove
tubes.
Do not open the lid until rotor stops.
23
SOP CEN-002
USE OF EPPENDORF 5804R CENTRIFUGE
Revision A, May 31, 2010
Title: Use and Maintenance of Eppendorf 5804R Tabletop Centrifuge
1.0 SCOPE AND APPLICATION:
This SOP outlines the use and maintenance of the Eppendorf 5804R refrigerated centrifuge.
2.0 SUMMARY OF METHOD
PROCEDURE
COMMENTS
1. Turn centrifuge on with main power
switch (on right side of unit).
2. Press the power button (if off, it will be lit
red; if on, it is lit green).
3. Press the LID button to open the
centrifuge lid, if it is not already open.
4. Determine the correct rotor for your
application. If it is installed, proceed to
next step. If not installed, see Appendix
for instructions on switching out the
rotor.
5. Open the cover of the rotor by lifting
off or rotating and lifting, depending on
the design of the rotor.
6. Set temperature, time, and speed
values to the appropriate settings using
the cursor keys. To change speed units
from “rpm” to “rcf”, press the speed
button to change the symbol
displayed.
7. You may need to wait for the
centrifuge to cool to a chilled
temperature before starting. If so, close
the lid and then allow the centrifuge to
cool down before loading.
8. Load tubes or plates into centrifuge
rotor in a balanced configuration.
9. Replace rotor cover.
10. Close centrifuge lid and press down to
lock in place; you will hear a click.
Balance tubes and plates weighing an
equal amount on opposite sides of the
rotor. This will reduce the risk that the
centrifuge becomes unbalanced and
vibrating. Additionally, samples may be
thrown out if unbalanced, possibly causing
damage to the centrifuge and loss of
samples.
24
11. Press the START key to begin the run.
12. If needed, press the STOP key to stop
the run before the elapsed time. Allow
the rotor to slowly decrease speed.
13. Press LID to open the lid and remove
tubes or plates.
14. Replace the rotor cover and turn off
centrifuge when finished.
15. It is best to leave the centrifuge lid
open when not in use, to prevent
microbial growth inside the chamber.
APPENDIX: Changing rotors
1. Remove the lid of the rotor.
2. Insert rotor key (simply a hex/Allen
wrench with handle) into the top of the
rotor and rotate to unfasten.
3. Lift the rotor out of the centrifuge. The
rotor may stick; wiggle the rotor while
firmly lifting to unstuck.
4. Place the new rotor in the centrifuge,
aligning the center with the post in the
middle of the centrifuge.
5. Use the rotor key to fasten the rotor
down.
6. Store the extra rotor safely, preferably
inside a padded box.
Do not open the lid until rotor stops.
25
SOP CEN-003
VWR CLINICAL 200 CENTRIFUGE
Revision A, May 31, 2011
Title: Use and Maintenance of VWR Clinical 200 Centrifuge
1.0 SCOPE AND APPLICATION:
This SOP outlines the use and maintenance of the VWR Clinical 200 centrifuge.
2.0 SUMMARY OF METHOD
PROCEDURE
COMMENTS
1. Turn centrifuge on with main power
switch (on left underside of unit).
2. Press the OPEN button to open the
centrifuge lid, if it is not already open.
3. Load 15-mL tubes into centrifuge rotor
in a balanced configuration.
4. Close centrifuge lid and press down to
lock in place; you will hear a click.
5. Set the speed by pressing the rpm/rcf
key and using the up and down arrows
to set the speed. To switch between
the units of speed, press the rpm/rcf
key twice.
Balance tubes weighing an equal amount
on opposite sides of the rotor. This will
reduce the risk that the centrifuge
becomes unbalanced and vibrating.
Additionally, samples may be thrown out if
unbalanced, possibly causing damage to
the centrifuge and loss of samples.
6. Set the time by pressing the time key
and using the up and down arrows.
7. Press the START key to begin the run.
DO NOT OPEN THE LID UNTIL ROTOR STOPS.
8. If needed, press the STOP key to stop
the run before the elapsed time. Allow
the rotor to slowly decrease speed.
9. Press LID to open the lid and remove
tubes.
10. Turn off centrifuge when finished.
11. It is best to leave the centrifuge lid
open when not in use, to prevent
microbial growth inside the chamber.
26
SOP CEN-004
USE OF VWR GALAXY 16D MICROCENTRIFUGE
Title: USE AND MAINTENANCE OF VWR GALAXY 16D MICROCENTRIFUGE
1.0 SCOPE AND APPLICATION:
This SOP outlines the use and maintenance of the VWR Galaxy 16D microcentrifuge.
2.0 SUMMARY OF METHOD
PROCEDURE
COMMENTS
1. Turn centrifuge on with main power
switch (on back of unit).
2. Lift lid to open.
3. Load 1.5-2.0 mL tubes into centrifuge
rotor in a balanced configuration.
Replace rotor cover.
4. Close centrifuge lid.
5. Set the speed using the up and down
arrows next to the speed display, up to
14,000 rpm. Toggle between rpm and
g-force using the mode key.
Balance tubes weighing an equal amount
on opposite sides of the rotor. This will
reduce the risk that the centrifuge
becomes unbalanced and vibrating.
Additionally, samples may be thrown out if
unbalanced, possibly causing damage to
the centrifuge and loss of samples.
6. Set the time using the up and down
arrows next to the time display (in
minutes).
7. Press the START key to begin the run. Lid
will lock during the run and will not
open until the rotor stops.
8. If needed, press the STOP key to stop
the run before the elapsed time. Allow
the rotor to slowly decrease speed.
9. The lid will unlock and the unit will beep
when the run is finished.
10. Turn off centrifuge when finished.
DO NOT OPEN THE LID UNTIL ROTOR STOPS.
27
SOP CUV-001
SELECTING, USING, AND CLEANING CUVETTES
Revision A, March 31, 20
Title: Selecting, Using and Cleaning Cuvettes
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for selecting, using and cleaning cuvettes.
1.0 INTRODUCTION AND SELECTION OF CUVETTES
Cuvettes are used as containers for spectroscopic and fluorometric readings and as such, must
be optically clear and free from scratches and chemical contamination.
Cuvettes can be made of a variety of materials, including glass, quartz, and plastic, and vary
widely in quality, cost, and versatility.
Materials
The most expensive cuvettes are made of quartz and have very transparent optical qualities.
These cuvettes are sometimes made in matched sets that can be used for a blank and a
sample to minimize the error contributed by a difference in optical properties between
cuvetttes. These are used for highly sensitive assays that are subject to error.
Glass cuvettes are often used for visible range spectroscopy because high-quality glass is
optically clear in the visible range.
Plastic cuvettes are very inexpensive and are usually disposable. However, one must be very
careful when selecting a plastic cuvette because many plastics are not transparent to UV light
and also can autofluoresce, making them unsuitable for UV spectroscopy and fluorescence
assays.
Design
For fluorescence, it is important that all four sides of the cuvette are optically clear because the
excitation light will pass through one window, while the emission leaves at a 90º angle to it. This is
unlike a spectrophotometry cuvette which needs to allow light to pass through in a straight line.
Some spectrophotometry cuvettes are designed for very small samples, often as small as 50 uL.
This allows the technician to load a small amount of a precious sample without having to dilute it
to fill the cuvette. These are sometimes referred to as microcuvettes. Cuvettes designed like this
are sometimes supplied sterile and nuclease-free so that the sample can be recovered without
becoming contaminated.
If the viewing window is isolated (i.e., the entire side of the cuvette is not transparent and there
are some frosted areas), it is important that the light beam height is compatible with the viewing
window height. In other words, the light beam must be able to pass through the viewing
window. Sometimes the beam height of the instrument is higher or lower than the viewing
window of the cuvette you have available. If the beam height is too low, nothing can be done
and you will need to find another cuvette. However, if the beam height is too high, you may be
able to insert a height adaptor into the sample compartment that brings the viewing window to
the correct height.
Note: Microplates used for optical experiments in a microplate reader can be selected using
much of the same criteria as above. Microplates are all plastic and so the optical clarity and
fluorescence properties must be considered when selecting a plate. Fluorescence assays are
often done using opaque white or black plates, which reduces the transfer of signal between
wells.
28
2.0 SUMMARY OF METHOD
PROCEDURE
2.1 USING CUVETTES
1. Select an appropriate type of cuvette
for the optical method and instrument
you need to use. Consider the material
and design of the cuvette when
making your selection (see
introduction).
2. If using previously used cuvettes, make
sure that they are clean inside and free
from water spots or scratches,
especially where the light beam will
pass through.
3. Fill cuvettes with adequate liquid so
that the light beam will pass through
the liquid. This can vary widely
depending on the cuvette design.
Usually the package will define the
minimum sample volume.
4. Wipe the viewing sides of the cuvette
with a lint-free wipe before loading into
the sample compartment to remove
fingerprints or dust. Always handle
cuvettes with gloved hands to minimize
fingerprints and hold cuvettes away
from the viewing windows.
2.2 CLEANING CUVETTES
1. Cuvettes to be reused should never be
allowed to dry out because this leaves
a residue that is very hard to clean off.
Sink used cuvettes in a container of
deionized water immediately after
using. Leave to soak for no more than
24 hours before draining out the water.
2. Rinse the inside of each cuvette with
70%. Use a squirt bottle.
3. Rinse each cuvette thoroughly with
deionized water.
4. Place cuvettes upside down on a test
tube drying rack or on top of an
absorbent towel. Do not leave the
COMMENTS
29
cuvettes to dry inside a beaker or other
deep container, since they will take too
long to dry and will also retain dried
residue from the water.
5. Reuse disposable glass and plastic
cuvettes no more than three times
before discarding. Quartz cuvettes can
be reused again and again until they
break or are badly scratched.
30
SOP DTR-001
DIGITAL TEMPERATURE RECORDER
Revision A, June 8, 2011
Title: Use and Maintenance of Digital Temperature Recorder
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for use of a digital temperature recorder.
2.0 SUMMARY OF METHOD
MATERIALS
1. Digital temperature recorder
2. Probe
PART A: OPERATION
1. Insert the temperature probe into the
recorder.
2. Press the round yellow power button on
the recorder to operate. Timer will start
when unit is turned on.
3. Situate probe in the area in which you
want to measure temperature. Close
the door to freezers, incubators, etc, on
the cord gently so you do not damage
the cord.
4. Allow the probe to equilibrate to the
temperature. This may take a few
minutes.
5. Record temperature for the desired
time period.
6. Remove probe and turn off recorder.
31
SOP ELC-002
MINI-PROTEAN CELL
Revision A, February 22, 2011
Title: Operation of Bio-Rad Mini-Protean Vertical Electrophoresis System
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for operation of the Bio-Rad Mini-Protean vertical
electrophoresis system (“cell”).
2.0 INTRODUCTION
The Mini-Protean cell is designed for fast and simple vertical electrophoresis using
polyacrylamide gels. Precast Bio-Rad Ready Gels are recommended; handcast gels require a
casting apparatus that we do not have in our lab. One or two gels can be run at a time.
3.0 SAFETY
Do not operate the Mini-Protean Cell at a voltage higher than 600 volts.
Before use, inspect the tank for cracks or chips, which may allow the buffer to leak from the tank
and cause a potential electrical hazard. Additionally, inspect all electrical cables, banana
jacks, and plugs for loose connections, cracks, breaks, or corrosion. Do not use any part that is
cracked, charred, or corroded. These parts may also cause a potential electrical hazard.
Contact your local Bio-Rad representative before using a part that may be considered
hazardous.
During electrophoresis, inspect the base and workbench for any signs of buffer leakage.
If leaking buffer is detected, disconnect the power to the cell immediately and contact your
local Bio-Rad representative.
4.0 SUMMARY OF METHOD
MATERIALS
1. Mini-Protean Cell (mini tank,
assembly, and lid)
2. Precast polyacrylamide gel
3. 150V power supply
4. 1X gel running buffer
5. Sample loading buffer
6. Samples to be loaded
7. Micropipettor and gel loading tips
PART A: GEL PREPARATION AND ASSEMBLY
1. Remove the Ready Gel from the
storage pouch.
2. Gently remove the comb. Pull off the
tape at the bottom of the Ready Gel
cassette to expose the bottom edge of
the gel.
3.
Set the clamping frame to the open
32
position on a clean flat surface.
4. Place the first gel sandwich or gel
cassette (with the short plate facing
inward) onto the gel supports; gel
supports are molded into the bottom of
the clamping frame assembly; there
are two supports in each side of the
assembly.
5. Now, place the second gel on the
other side of the clamping frame,
again by resting the gel onto the
supports.
6. Using one hand, gently pull both gels
towards each other, making sure that
they rest firmly and squarely against the
green gaskets that are built into the
clamping frame; make certain that the
short plates sit just below the notch at
the top of the green gasket.
It is critical that gel cassettes are placed
into the clamping frame with the short
plate facing inward. Also, the clamping
frame requires 2 gels to create a
functioning assembly. If an odd number of
gels (1 or 3) is being run, you must use the
buffer dam.
7. While gently squeezing the gel
sandwiches or cassettes against the
green gaskets with one hand (keeping
constant pressure and both gels firmly
held in place), slide the green arms of
the clamping frame over the gels,
locking them into place.
8. The arms of the clamping frame push
the short plates of each gel cassette up
against the notch in the green gasket,
creating a leak-proof seal (check
again to make certain that the short
plates sit just below the notch at the
top of the green gasket). At this point,
the sample wells can be washed out
with running buffer, and sample can be
loaded.
PART B: RUNNING THE GEL
1. Place the assembled gel(s) into the
tank. The assembly should be inserted
into the tank on the side away from
you, with the black electrode on the
left.
2. Pour 1X running buffer into the
The lid only goes on one way, so it is
important to insert the assembly correctly.
You do not want to have to move it after
the gels are loaded with samples.
33
assembly (upper chamber). Before
filling the rest of the tank, make sure the
assembly does not leak. If it does, you
must remove the assembly and
assemble it correctly.
3. Fill the upper chamber with buffer until
the level is just below the outer plate(s)
of the gel(s). Fill the tank up to the 2-gel
level marker (requires about 700 mL).
4. Make sure the wells are all filled with
buffer. Use a pipet to blast out any
stubborn air bubbles. This step also
removes unpolymerized acrylamide
from the wells, which can interfere with
protein migration.
5. Load samples and standards into the
wells using a gel loading pipet tip or
syringe. Be careful not to poke a hole in
the gel or blast samples out when
loading (stop just short of the second
stop).
6. Place the lid on the base, matching the
electrode colors.
7. Connect the electrical leads to a
power supply. Set to 200 volts and press
start.
8. Check the area around the electrodes
to make sure tiny bubbles are forming.
This is evidence of an electrical current
in the buffer.
9. Periodically check the system to make
sure the tracking dye is migrating in the
gel. Do not allow the tracking dye to
migrate off your gel. Do not allow the
buffer to heat above 40ºC or your gel
could melt.
10. When you are finished running the gel,
stop the power supply and disconnect
the leads.
11. Remove the lid from the base and take
out the assembly. Pour the buffer out
before disassembling.
Be careful not to jostle or move the base
after loading wells because you will lose
your sample(s).
34
12. Remove the gel from the assembly.
13. Remove the gel from its cassette by
separating the two plates of the
cassette. This can be done easily by
inserting a spatula between the plates
and twisting.
14. Remove the top plate and invert the
other plate in a container of water,
buffer, or staining solution. Agitate
gently until the gel falls off the plate.
15. Fix and/or stain the gels as desired.
16. Visualize the gel using the appropriate
light source (white light for Coomassie
blue stain).
17. Discard the electrophoresis buffer and
gel.
18. Rinse the lid, tank, and assembly with
tap water and dry completely before
storing.
Be extremely careful with polyacrylamide
gels, which are very fragile and rip easily.
35
SOP ELC-003
MINI TRANS-BLOT TRANSFER CELL
Revision A, June 8, 2011
Title: Operation of Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for operation of the Bio-Rad Mini Trans-Blot
Electrophoretic Transfer Cell.
2.0 INTRODUCTION
The Mini Trans-blot cell is part of Bio-Rad’s modular Mini-Protean Tetra system for electrophoresis
and blotting. The cell fits into the Mini-Protean tank and can be used for electrophoretic transfer
of species from a gel onto a membrane.
3.0 SAFETY
Before use, inspect the tank for cracks or chips, which may allow the buffer to leak from the tank
and cause a potential electrical hazard. Additionally, inspect all electrical cables, banana
jacks, and plugs for loose connections, cracks, breaks, or corrosion. Do not use any part that is
cracked, charred, or corroded. These parts may also cause a potential electrical hazard.
Contact your local Bio-Rad representative before using a part that may be considered
hazardous.
During operation, inspect the base and workbench for any signs of buffer leakage.
If leaking buffer is detected, disconnect the power to the cell immediately and contact your
local Bio-Rad representative.
4.0 SUMMARY OF METHOD
MATERIALS
1. Mini-Protean tank and lid
2. Mini Trans-blot cell
1. Electrode module
2. Gel holder cassette
3. Fiber pads (2)
4. Cooling unit
3. Filter papers (2)
4. Membrane
5. Gel containing separated samples
6. Roller or glass test tube
7. 200V power supply
8. 1X transfer buffer (refrigerated)
PART A: PREPARATION FOR BLOTTING
1. The cooling unit should be filled with
water and frozen before use. Chilling
the transfer buffer before using will also
help maintain a cool temperature
during the transfer.
36
2. Cut the membrane and filter paper to
the dimensions of the gel, if necessary.
3.
Equilibrate the gel and soak the
membrane, filter paper, and fiber pads
in transfer buffer (15-20 minutes).
Always wear gloves while handling
membranes.
Be extremely careful with polyacrylamide
gels, which are very fragile and rip easily.
4. Prepare the gel sandwich:
a. Place the cassette, with gray
side down, on a clean surface.
b. Place one pre-wetted fiber pad
on the gray side of the cassette.
c. Place a sheet of filter paper on
the fiber pad.
d. Place the equilibrated gel on
the filter paper.
e. Place the pre-wetted
membrane on the gel.
f. Place a piece of filter paper on
the membrane.
g. Place a fiber pad on the filter
paper.
Remove any air bubbles under the gel or
membrane by rolling them out. Use a
specialized roller or simply a glass test tube.
5. Close the cassette firmly, being careful
not to move the gel and filter paper
sandwich. Lock the cassette closed
with the latch.
6. Place the assembled cassette into the
module.
7. Place the module into the tank with the
black electrode to the top and left.
Add a cooling unit to the tank. Fill the
tank with buffer to the “blotting” mark
on the tank.
8. Add a small stir bar to the tank and
place the tank on top of a stir plate. Set
the stirring speed as fast as possible
without splashing.
Stirring the buffer will help maintain a cool
and even buffer temperature and
homogeneous ion concentration.
9. Place the lid on the tank, matching the
electrode colors.
The lid only fits on one way, so make sure
the module is oriented correctly.
37
PART B: RUNNING THE GEL
1. Connect the electrical leads to a
power supply. Set to an appropriate
voltage and current and run the
transfer.
2. After starting, check the area around
the electrodes to make sure tiny
bubbles are forming. This is evidence of
an electrical current in the buffer.
3. At the end of the run, turn off the
power supply and disconnect the
leads.
4. Remove the lid from the tank. Remove
the module from the tank and the
cassette(s) from the module.
5. Disassemble the cassette(s) and set
aside the membrane. Discard the
transfer buffer, filter papers, and gel.
6. Detect species on the membrane using
the appropriate detection method.
7. Rinse the lid, tank, fiber pads, cassettes,
and module with tap water and dry
completely before storing.
The voltage, current, and time should be
selected based on the type of buffer, gels,
and analyte(s). One option for SDS-PAGE
gels containing proteins in TG buffer with
alcohol would be a one-hour transfer at
100 V and 250 mA.
38
SOP ELC-004
EDVOTEK ELECTROPHORESIS CHAMBERS (M12 AND HEXAGEL)
Revision A, June 25, 2012
Title: Operation of Edvotek Horizontal Electrophoresis Chambers
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for operation of the Edvotek M12 and Hexagel
horizontal electrophoresis chambers.
2.0 INTRODUCTION
The Edvotek system is designed for fast and simple horizontal electrophoresis using agarose gels.
The gel is cast outside the base chamber using the included tray, combs, and bumpers for
casting.
3.0 SAFETY
Do not operate the apparatus at a voltage higher than 300 volts.
Before use, inspect the box base for cracks or chips, which may allow the buffer to leak from the
base and cause a potential electrical hazard. Additionally, inspect all electrical cables, and
plugs for loose connections, breaks, or corrosion. Do not use any part that is cracked, charred, or
corroded. These parts may also cause a potential electrical hazard. Contact your laboratory
technician.
During electrophoresis, inspect the base and workbench for any signs of buffer leakage.
If leaking buffer is detected, disconnect the power to the cell immediately and contact your
laboratory technician.
4.0 SUMMARY OF METHOD
MATERIALS
1. Edvotek electrophoresis chamber
(M12 or Hexagel)
2. Gel casting equipment
1. Bumpers (2 per tray)
2. Casting tray (7x7 cm or
7x14cm)
3. Comb (6, 8 or 10 well)
3. Electrophoresis power supply
4. Agarose powder
5. 1X Running buffer such as TAE or TBE
6. Sample buffer/loading dye
7. Samples to be loaded
8. Micropipettor and tips
39
PART A: GEL CASTING
1. Prepare 1X buffer and molten agarose.
The 7x7cm casting trays hold
approximately 30mL of molten agarose,
and the 7x14cm hold about 60mL.
2. Cool the molten agarose to 50-60ºC.
3. Press the black bumpers onto either
side of the casting tray to form a tight
seal. This is easiest if you place the
bumper slit up on the bench and press
the tray down onto it.
Warning: Hot agarose (>60°C) may cause
the tray to warp or craze and will decrease
the lifetime of the tray. Warping may also
result in sample wells of uneven depth.
4. Select a comb (blue or white) with the
appropriate number of wells, and insert
it into the tray.
5. Pour about 30mL of agarose into the
7x7 tray (60mL into the 7x14.) Check for
bubbles or debris and remove these
with a transfer pipet before the
agarose cools.
6. Allow 20-40 minutes for the agarose to
cool and solidify.
7. Carefully remove the comb and
bumpers from the gel.
To avoid ripping the gel, it can be helpful
to separate the gel from the bumpers and
tray using a slim spatula before attempting
to remove.
PART B: RUNNING THE GEL
1. Place the casting tray with solidified gel
into the electrophoresis chamber,
orienting the wells toward the black
electrode. The tab on the side of the
tray will fit into a cut out on the side of
the chamber.
2. Pour 1X electrophoresis running buffer
over the gel until it is covered by 2-6
mm.
3.
Make sure the wells are all filled with
buffer. Use a pipet to blast out any
stubborn air bubbles.
4. Load samples and standards (premixed
with sample buffer/loading dye to a
final concentration of 1X) into the wells
using a regular pipet tip. Be careful not
to poke a hole in the gel or blast
samples out when loading (stop just
short of the second stop).
The M12 chamber requires about 400 mL
buffer, while the Hexagel needs 2-3 times
that amount.
40
5. Place the lid on the base, matching the
electrode colors (black to black and
red to red.)
6. Connect the electrical leads to a
power supply and choose an
appropriate voltage, usually between
75 and 150 volts. Press start.
Be careful not to jostle or move the base
after loading wells because the sample(s)
can float out.
Edvotek DuoSource power supplies can be
set to either 75 or 150 volts. Other power
supplies can be adjusted to more specific
voltages.
7. Check the area of buffer around the
electrodes to make sure tiny bubbles
are forming. This is evidence of
electrical current passing through the
buffer.
8. Periodically check the system to make
sure the tracking dye (in the sample
buffer) is migrating down the gel. Do
not allow the tracking dye to migrate
off your gel. Do not allow the buffer to
heat above 40ºC or your gel could
melt. If the chamber is very warm to the
touch or excessively steamy, you may
be overheating the system.
Voltage is directly related to heat. If your
gel is becoming hot, reduce the Voltage
so as not to melt the gel.
9. When you are finished running the gel,
stop the power supply and disconnect
the leads.
10. Remove the lid from the base. Carefully
take out the gel in the tray and place it
in a container for transport to the gel
documentation system.
11. Discard the electrophoresis buffer and
gel, treating as hazardous waste if
necessary.
12. Rinse the lid, base, tray, comb, and
bumpers with tap water and dry
completely before storing. If agarose
residue remains on any of the casting
parts, wash gently with soapy water
and a brush, rinse thoroughly, and dry.
Avoid scrubbing or aggressively drying the
inside of the chamber because the
platinum electrodes are delicate and can
be ripped out easily. A gentle tap water
rinse and blot-dry with a paper towel is
sufficient and will not damage the
electrodes.
41
SOP ERP-001
USE OF EPPENDORF REPEATER PLUS PIPETTOR
Title: Operation and maintenance of Eppendorf Repeater Plus Pipettor
1.0 SCOPE AND APPLICATION:
This SOP outlines the procedure for operation and maintenance of the Eppendorf Repeater Plus
pipettors.
2.0 INTRODUCTION
The Repeater Plus forms a dispensing system for repetitive pipetting when used in conjunction
with Eppendorf Combitip Plus Tips. A supply of liquid is aspirated into the Combitip Plus and then
dispensed step by step. The built-in electronics of the device automatically recognize the
Combitip Plus inserted and the position of the volume selection dial, and the current pipetting
volume appears in the display. Using the volume selection dial, 20 different volumes can be
selected for each Combitip Plus. The smallest pipetting volume can be dispensed a maximum of
100 times. The largest pipetting volume can be dispensed a maximum of 5 times.
3.0 SUMMARY OF METHOD
PART A: OPERATION
COMMENTS
1. Determine the volume you would like
to dispense, and choose a Combitip.
2. Insert the Combitip into the pipettor
from the bottom; it will click into place.
25- and 50-mL tips require an adaptor.
3. The selected volume will appear on the
display. To change the selected
volume, rotate the dial left or right.
4. Push the filling lever all the way down.
5. Insert the tip into the liquid to be
dispensed. Pull the filling lever up all the
way while keeping the tip submerged.
6. Press the pipetting lever once to
discard the first measurement (not
accurate).
7. Press the pipetting lever and the
correct volume will be dispensed.
8. Refill tip if necessary by repeating Step
5.
9. When you are finished dispensing,
discard any leftover fluid by pressing
Always keep the micropipettor in vertical
position to ensure that no liquids enter the
micropipetter or drip from the disposable
tip.
42
filling lever all the way down.
10. Eject tip by pressing on the ejection
keys at the sides of the pipette.
43
SOP FLU-002
VARIAN CARY ECLIPSE FLUOROMETER
Revision A, June 8, 2011
Title: Operation of the Varian Cary Eclipse Fluorometer
1.0 SCOPE AND APPLICATION:
This SOP outlines the procedure for operation of the Varian Cary Eclipse fluorometer.
2.0 INTRODUCTION
The Cary Eclipse fluorescence spectrophotometer can read fluorescence intensity at defined
wavelengths, scan a sample for emission and excitation maxima, perform kinetics assays, and
can also be used to read phosphorescence and chemiluminescence. The xenon flash lamp is
long-lasting and never needs replacing; excitation and emission wavelengths are tunable to 1
nm. The instrument is operated through an attached computer with Eclipse software.
3.0 SUMMARY OF METHOD
COMMENTS
PART A: MATERIALS
1. Varian Cary fluorometer
2. Laptop computer with Windows XP
OS and Eclipse software installed
3. Fluorometry cuvettes (optically clear
on all four sides)
The Eclipse software has not been tested
with Windows 7, so be sure your attached
computer has Windows XP.
PART B: SCANNING
For determining emission and excitation
maxima for an uncharacterized
sample, you will want to use the Scan
module.
1. Turn on the fluorometer using the on/off
switch on the bottom front of the unit.
A yellow LED will come on while the
instrument calibrates; wait about 2
minutes until calibration is over and the
light will turn green.
2. Connect the flurometer to a laptop via
USB cable and turn on the computer.
Click on the Cary Eclipse folder on the
desktop and then select the Scan icon.
3. Insert sample. Open the sample
compartment by sliding the lid back.
Place a fluorometry cuvette containing
your sample in the compartment and
close the lid.
A cuvette with frosted sides will not work
because the light paths in and out of the
cuvette are at a right angle. All four sides
must be optically clear and made of lowfluorescence plastic.
44
4. Select Open Method from the File
menu to load a previously defined
method. If you don’t have a method,
click the Setup button. Select the
Excitation or Emission button, and enter
wavelengths for excitation and
emission and the scan speed. Select
the Options tab, and select PMT
voltage. Start with Medium. Click OK.
5. When you have loaded a previous or
new method, you will see the main
screen. Click on the traffic light icon
(“Start”) to begin reading your sample
using the selected method. You will be
prompted to name your sample; do so
and click OK. The scan will begin and
you will see the wavelength changing
in the upper right corner.
6. When the scan is complete you will see
a fluorescence spectrum.
7. If spectrum intensity is weak or clipped,
go back to Setup and adjust PMT
voltage and any other parameters
desired. Higher PMT voltage and larger
slits give higher sensitivity.
8. Remove the sample from the
compartment. Load another sample
and repeat the process.
9. When finished, remove the last sample
from the compartment and close the
lid. Turn off the fluorometer and the
computer. Rinse your cuvettes with
deionized water and/or alcohol and
place upside down to dry.
45
SOP FPLC-001
BIO-RAD DUOFLOW FPLC SYSTEM
Revision B, April 25, 2011
Title: Use and Maintenance of the Bio-Rad DuoFlow Fast Liquid Chromatography System
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for use and maintenance of the Bio-Rad DuoFlow
Forward Phase Liquid Chromatography (FPLC) System.
2.0 INTRODUCTION
The Bio-Rad DuoFlow FPLC system offers a simple yet versatile way to do liquid chromatography.
This system is equipped to perform forward phase liquid chromatography at medium pressure.
The system consists of several components:
 Workstation with dual-piston F10 pumps
 Maximizer mixer
 AVR7-3 automated injection valve
 Dual wavelength UV detector
 Conductivity and pH monitors
 BioFrac fraction collector
These components can be operated manually or controlled using the BioLogic software on an
attached computer. This SOP will describe how to operate using BioLogic.
The DuoFlow F10 pumphead operates at up to 20 mL/min and 3500 psi when used with the
Maximizer.
3.0 SUMMARY OF METHOD
MATERIALS
1. FPLC system (list of components in
Introduction)
2. Filtered and degassed buffer and
salt solutions and water
3. Injection syringe and flat tipped
needle (both Luer-lok)
4. Chromatography column
5. Sample
6. Waste container (large beaker,
flask, or bottle)
PART A: STARTUP
1. Select the appropriate column, buffers,
and salt solution for your analysis. Filter
(to at 0.2 µm) and degas buffers and
deionized water.
2. Turn on the Maximizer, workstation
pumps, and fraction collector using the
power toggle switches on the front or
back of each component.
46
3. Open the BioLogic software on the
attached computer.
4. When the system is first started up for
the day or after a long storage, you
must prime the pumps in order to
remove any air.
5. Make sure that the column is not in-line
before proceeding! The column is
installed after the sample inject valve
and before the UV flow cell.
6.
Submerge all four inlet tubing lines in
pure water. Secure all three waste lines
(narrow, translucent white tubing) in a
large waste container.
Check the manual screen for the following
system components: Maximizer and
gradient pump, fraction collector, UV
detector, and workstation valve AVR7-3.
You may need to click the arrow at the top
right of the valve window to view the
AVR7-3 valve.
The column may become damaged from
high pressure during priming or purging, so
it should be removed from the line before
these steps.
7. In the software, place the Maximizer in
Local mode (so you can control it
manually).
8. Attach a large Luer-lok syringe (20mL or
larger) to the port on pump A. Twist left
and draw out the syringe. The first pull
may just be air. Twist the port closed
(with syringe attached!) and then
remove the syringe and expel air and
liquid. Repeat once or twice more until
the syringe contains mostly liquid.
9. Repeat step 5, but this time, press the
port select button below to select A2.
10. Repeat steps 5 and 6 on pump B and
make sure to prime both B1 and B2
using the port select button.
11. The next setup step is to purge the
system. In this step, you flush the entire
system with clean eluent. This is
especially important if you had the
system stored in ethanol or sodium
azide. In this case, you should flush with
water only to avoid salt precipitation.
12. In the software, place the Maximizer in
System mode so you can control using
BioLogic. Locate the AVR7-3 valve and
click “P” to place the valve in the
Purge position (you will hear the valve
If you remove the syringe before closing
the port, air will enter the pump.
47
move).
13. Place the Maximizer in Local mode
again. Press the Purge buttons A and B
on the front of the pump (lights will flash
green). Wait two minutes, then press
the valve port select buttons on the
front of the Maximizer to select the
other two ports. Purge those for two
minutes more. Press the purge buttons
to stop the pump.
14. Place the Maximizer in System mode
and the AVR7-3 valve in the Load
position by clicking “L”. Set the pump
flow rate at 1.0 mL/minute and start the
pump. Water will flow through the
entire system and out of the fraction
collector.
15. Before the first run of the day, calibrate
the pH electrode. Unscrew the pH
monitor to expose the electrode and
place it in pH 7 standard buffer.
16. From the Utilities menu, select pH probe
calibration. Press Set. When the reading
has stabilized, click OK.
17. Rinse the electrode with deionized
water.
18. Repeat with pH 10 standard buffer.
19. Reassemble the pH monitor.
20. Before the first run can be initiated, the
system and column should be
equilibrated.
21. Submerge the inlet tubing lines in the
appropriate buffer(s), salt, and water.
a. A1 (red): acid (such as Tris-HCl)
b. A2 (blue): base (such as Tris
base)
c. B1 (yellow): water
d. B2 (green): salt
22. Prime and purge the pumps as before.
23. Place the column in line. First remove
48
the union that connects the line, then
remove the end caps from the stored
column and install where the union
was, making sure you orient it so the
flow will pass through in the direction of
the arrow on the column.
24. In the Manual screen of the software,
set the pressure limits to the
recommended values for the column.
25. Set the inject valve position to Load (L).
Set the flow rate to 2.0 mL/minute. Set
the buffer recipe by pressing the Setup
button on the toolbar. Select the
appropriate buffer pH and salt
concentration for your protocol.
26. Wash the column with equilibration
buffer conditions and 100% B (highest
possible salt concentration) for about 3
minutes.
27. Equilibrate the column with the same
buffer but change to 0% B for about 7
minutes.
28. The system is now ready to run a
separation.
PART B: OPERATION
1. Open the Browser tab to locate a
saved method, filed under different
Users. Double-click the method and the
protocol will open.
2. Alternatively, create a new method.
Click New Method on the toolbar and
you will be prompted to pick a user
and name the method.
3. A method editor will open. On the left
side of the screen you will be able to
add instrument components to the
method. These should be added:
 Workstation
 Mixer
 AVR7-3 valve
 UV detector
 Conductivity
Setting the upper pressure limit will protect
the column from excessive pressure that
could damage it.
49


pH monitors
Fraction collector
4. You also need to design a protocol for
the method. Click the protocol icon on
the toolbar and an editor will open.
Add steps such as turning on the UV
lamp, zero baseline, isocratic flow,
inject sample, and linear gradient to
create your protocol. Make sure to
equilibrate before the injection and
after the run is over.
5. When you have created and/or
opened the method you need, you
may begin. Click Run on the toolbar
and the Run screen will appear.
6. Load sufficient 12 mm tubes into the
fraction collector.
7. Place the inject valve in the Load (L)
position. Draw up a volume at least 10%
more than the sample loop installed
into a Luer-lok syringe with a flat-tipped
needle.
8. Place the needle in the inject port and
fill the loop, injecting more than the
loop volume to rinse it out. Do not
remove the needle.
9. Click the green Start button on the
toolbar to launch the run. The sample
will be injected automatically at the
correct step in the protocol.
10. As the run progresses, each step in the
protocol will acquire a checkmark as it
is completed. The Run screen displays
three traces:
a. UV absorbance—shows species
as they leave the column
b. Conductivity—tracks the
progress of a salt gradient
c. pH—tracks the progress of a pH
gradient
11. When the run is finished, the pumps will
automatically stop and a Run Finished
message appears at the bottom right
50
of the status bar.
PART C: SHUTDOWN
1. After the last run of the day, make sure
that no sample is left in the injector or
column. Each method should include a
step that washes sample off the
column so it is clean for the next run, as
well as a final equilibration step. If this is
done with the last run of the day, there
is no need to further wash the system.
High salt buffer should not be left in the
system overnight.
2. Wash behind each pumphead with
about 15 mL of water.
3. If the instrument is to be stored longer
than one week without using, you must
flush all buffers out of the system and
replace with an antimicrobial solution.
4. First, remove the column and replace
with a union. Inject the appropriate
antimicrobial solution through the
column using a Luer-Lok syringe and
cap both ends (see column
manufacturer instructions).
5. Place the inlet tubing into filtered,
degassed deionized water. Pump
water through the system at 2
mL/minute for about 5 minutes.
6. Wash behind each pumphead with
about 15 mL of water.
7. Transfer the inlet tubing into filtered 20%
ethanol or 0.05% sodium azide and
pump through the system at 2
mL/minute for about 5 minutes.
8. When the system is full of antimicrobial
solution, remove all eluent bottles and
turn off each component.
PART D: TROUBLESHOOTING
If you see a sawtooth pattern in the pump
pressure trace, you most likely have air
bubbles in the system. This is due to the
51
compressability of air compared to water.
Purge your pumps and start over.
52
SOP GDS-003
CHEMIDOC GEL DOCUMENTATION SYSTEM WITH QUANTITY ONE
Revision A, June 8, 2011
Title: Use of ChemiDoc Imaging System with Quantity One Software for Gel Documentation
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for using the Bio-Rad ChemiDoc imaging system with
Quantity One software for gel documentation.
2.0 SUMMARY OF METHOD
PART A: UV IMAGING OF ETBR- OR SYBR
SAFE-STAINED GELS
1. Turn on the system using the on/off
switches on the A/C power supply and
on the back of the unit.
To consult the instruction manual, doubleclick on the Quantity One Instruction
Manual icon on the desktop.
2. Open the software by double-clicking
on the Quantity One icon on the
desktop.
3. In the file menu, click on ChemiDoc
XRS. A new window will open. Under
Select Application, make sure that UV
Transillumination is listed as the
application. If it says something else,
such as “White Transillumination,” click
Select and choose UV Transillumination.
4. Position the gel(s) on top of the UV light
box and close the door and/or drawer
of the unit. Press the UV Trans button on
the front of the unit.
5. Click on Live View and an image of the
gel will appear. Change the iris, zoom,
and focus settings until a satisfactory
image is obtained. Click on Freeze. This
will activate the Acquire Image
options.
6. Click on Manual Expose. Wait a
moment for the image to appear in a
separate window. If the image is too
light or dark, or if there is saturation in
the image (shown in red), change the
exposure time setting and expose
again.
Reposition the gel in the drawer if
necessary. The UV light will automatically
switch off, and you will need to press the
UV Trans button when you close the
drawer.
If you make an exposure, the UV light will
53
7. If the image obtained is satisfactory,
you may save and print. To print, click
the print icon on the toolbar while the
image is in the active window. To save,
click the “X” in the top right corner of
the image window, then choose Save.
shut off. If you decide to make another
exposure, you will have to turn the UV light
back on by pressing the UV Trans button
again.
You must choose Mitsubishi P93D printer to
print to the thermal printer.
8. When you are finished imaging,
remove the gel from the drawer and
wipe the surface clean with a paper
towel. Turn the ChemiDoc off at both
power switches.
PART A: VISIBLE LIGHT IMAGING OF GELS
1. Turn on the system using the on/off
switches on the A/C power supply and
on the back of the unit.
Note: visible light imaging with the
ChemiDoc is currently done using a white
light converter screen on top of the UV
light box.
2. Open the software by double-clicking
on the icon on the desktop.
3. In the file menu, click on ChemiDoc
XRS. A new window will open. Under
Select Application, make sure that UV
Transillumination is listed as the
application. If it says something else,
such as “White Transillumination,” click
Select and choose UV Transillumination.
4. Place the white light converter screen
on top of the UV transilluminator inside
the cabinet.
5. Position the gel(s) on top of the
converter screen and close the door
and/or drawer of the unit. Press the
Trans UV button on the front of the unit.
6. Click on Live View and an image of the
gel will appear. Change the iris, zoom,
and focus settings until a satisfactory
image is obtained. Click on Freeze. This
will activate the Acquire Image
options.
7. Click on Manual Expose. Wait a
moment for the image to appear in a
separate window. If the image is too
light or dark, or if there is saturation in
the image (shown in red), change the
Reposition the gel in the drawer if
necessary. The UV light will automatically
switch off, and you will need to press the
UV Trans button when you close the
drawer.
54
exposure time setting and expose
again.
8. If the image obtained is satisfactory,
you may save and print. To print, click
the print icon on the toolbar while the
image is in the active window. To save,
click the “X” in the top right corner of
the image window, then choose Save.
9. When you are finished imaging,
remove the gel from the drawer and
wipe the surface clean with a paper
towel. Turn the unit off at both power
switches.
If you make an exposure, the UV light will
shut off. If you decide to make another
exposure, you will have to turn the UV light
back on by pressing the UV Trans button
again.
You must choose Mitsubishi P93D printer to
print to the thermal printer.
55
SOP GDS-004
CHEMIDOC IMAGER WITH IMAGE LAB SOFTWARE
Revision B, July 12, 2012
Title: Use of Bio-Rad ChemiDoc Imager Using Image Lab Software
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for use of the Bio-Rad ChemiDoc Imager with Image
Lab software for gel documentation.
2.0 SUMMARY OF METHOD
MATERIALS
1. ChemiDoc Imager
2. Attached computer with Image
Lab software
3. Gels to be imaged
PART A: OPERATION
1. Turn on the cabinet (switch is on the
back left of the cabinet) and the
camera power supply (black box).
2. Double-click the Image Lab icon on
the desktop to open the software.
3. You may use an existing protocol by
choosing Open under the File menu.
Proceed to step 9.
4. To create a new protocol, click New
Protocol on the menu bar. Under Gel
Imaging, select the application you
need (nucleic acid or protein gel) and
select the stain you are using.
5. You may also select a gel type from a
drop-down menu; this can be helpful if
you are using the ChemiDoc to analyze
your gel results. You may also enter the
image area manually.
6. You can select optimization for either
faint or intense bands, or you can
manually select the exposure time.
7. You may also select the color you want
the image displayed in, which should
be the same as your stain. Please note,
Important: the Mitsubishi P93D thermal
printer will only work on a computer using
Windows XP. Windows 7 will not allow the
printer driver to be installed. Image Lab
works fine with Windows 7, but images will
not print.
56
the printer will only print in black and
white.
8. If you would like to include an image
analysis and/or a report in your
protocol, check the boxes next to
these and select parameters for Lane
and Band Detection, Molecular Weight
Determination, and the type of output
for a report. These steps are optional.
9. When you are ready to run the
protocol, click the yellow Position Gel
button. Open the drawer on the
cabinet and place your gel on either
the UV light box or on top of the white
conversion screen (if you are using
white light illumination). For either UV or
white light (with the conversion screen),
you must have the filter in position 1.
10. Position the gel in the center of the box
and close the drawer. You may zoom
in or out by moving the camera meter
below the image. When the image
appears how you want it to, click the
green Run Protocol button. This will
initiate image collection.
11. After the exposure your image will
appear in a new window. Go to File >
Save to save the image. If you need to
open the file in another program such
as Microsoft Paint, you will need to
export the file as a JPEG. Do this by
going to File > Export > Displayed
Image and save the image as a JPEG.
12. To print, go to File > Print > and click
Print. Make sure the Mitsubishi P93D is
selected as the printer. This is a thermal
printer that uses glossy paper and prints
small images. When the image has
printed, pull against the cutting edge
to detach your photo.
13. When you are finished with the
ChemiDoc, wipe buffer and gel residue
off the light box with a paper towel.
Turn off the camera and the cabinet.
Another option is checking or unchecking
the box next to “Highlight Saturated Pixels.”
When checked, the display will show red
highlights over pixels that are saturated
(when the camera was at its max light
recording ability). These saturated pixels
indicate a high level of fluorescence or
intense color in that region of the gel.
Unchecking the box will hide the highlights.
57
PART B: TROUBLESHOOTING GUIDE
1. 1. If the ChemiDoc instrument is not
detected by the software, make sure
that BOTH the cabinet and the camera
power supply are turned on. Lights
should be visible on the front of the
cabinet if it is on.
2. 2. If you see a bright circle on the
image, check to make sure the flat
field lens is not in position 2 of the filter
wheel. This lens is white on top and pink
on the bottom and is placed in position
2 during a calibration step. It should be
removed after calibration, but if it was
mistakenly left on, it can cause a
circular reflection on gel images.
3. 3. If the printed images are an odd size
or shape, make sure the print size is
1280x1280. Go to File > Print > Page
Setup and choose 1280x1280 as the
page size.
58
SOP GWC-001
CLEANING GLASSWARE
Revision B, July 12, 2012
Title: Cleaning Glassware
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for cleaning used glassware and plasticware.
2.0 SUMMARY OF METHOD
PROCEDURE
COMMENTS
1. Remove any tape or labels from the
glassware and remove any pen marks
with acetone or ethanol.
Use of this SOP indicates that the user has
disposed properly of any hazardous
materials contained in the dirty glassware.
2. Fill a wash tub with warm tap water
and a few mL of Micro-90 detergent.
Use a flat razor scraper to remove tape
and labels, if necessary.
3. Soak dirty glassware for a few minutes
before scrubbing thoroughly with a
brush.
4. Rinse thoroughly with tap water three
times.
5. Rinse thoroughly with deionized water
three times.
6. To dry, hang glassware on pegboard if
possible, or place upside down over
paper towels or Labmat.
If a stubborn chemical residue persists, soak
the glassware in tap water and use a brush
or a spatula to remove the residue (only if
nonhazardous). Glassware with persistent
residue of a hazardous chemical should be
marked as hazardous waste (be sure to
name the chemical).
59
SOP HPLC-001
Waters Breeze HPLC System
Revision A, April 21, 2011
Title: Use and Maintenance of Waters Breeze HPLC System
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for use and maintenance of the Waters Breeze HPLC
System.
2.0 SUMMARY OF METHOD
MATERIALS
Waters Breeze HPLC System
PART A: OPERATION
1. Turn on the HPLC pump, column
heater, absorbance detector, and
degasser. Wait for them to initialize and
run self-diagnostic tests.
2. Turn on the computer attached to the
HPLC.
3. Open the Breeze software and allow it
to run through its system startup routine.
4. Minimize the Help window, if necessary.
The sample queue workspace should
be completely visible.
5. Click View Method on the command
bar. Click on the open icon to open a
method, or click the new icon to
create a new method.
6.
If you choose to create a new method
or edit an existing one, you will be able
to edit pump, heater, and detector
parameters by clicking on the images
of the pump and detector. These
parameters include pump flow rate,
isocratic or gradient flow type, column
heating, and detection wavelength(s).
7. When you have selected your desired
method, you can begin setting up the
instrument to perform a separation.
60
8. Submerge the vacuum degasser inlet
lines in the mobile phase. If your mobile
phase will be isocratic, submerge both
lines in the same buffer. If you are using
a gradient, submerge one line in each
of your buffers.
9. Click on the Purge Wizard button in the
acquisition bar at the bottom of the
window. The Purge Wizard dialog box
will open and “Purge Pump(s)” will be
checked. Click Next.
10. Prime the pumps. Open the front cover
of the binary pump and locate the
draw-off valves.
11. Insert a 20 mL Luer-lok syringe into the
inlet of a valve and rotate the valve
counterclockwise several turns to open.
Draw out the plunger of the syringe
until several mL of liquid is withdrawn.
With the syringe inserted, close the
valve, then remove the syringe and
empty it. Repeat this step if you see any
air bubbles in the inlet tubing for this
pump.
12. Repeat with the other draw-off valve.
13. Click Next on the Prime Pump dialog
box.
14. The Purge Pump dialog box appears.
The pump flow meters will be pre-set at
5 mL/minute.
15. Open the reference valve, located
near the draw-off valves for the pumps.
16. Click Next to proceed with purging the
pumps. This will last 5 minutes.
17. Click Next when the purge is complete.
Close the reference valve and click
Finish.
18. Equilibrate the system. Click the
Equilibrate button on the acquisition
bar. Select the appropriate method
from the drop-down list and click
Equilibrate.
61
19. Allow the system to equilibrate for 20
minutes, or until the baseline is stable.
20. Click on the Stop Pump button to stop
the equilibration.
21. Click on the Inject button to initiate a
run. You will be asked for the desired
method and information on the sample
to be injected.
22. The manual injection dialog box will
open. Fill a syringe with the sample and
insert into the manual injection port.
Inject the sample and using a fluid
motion, move the injection port lever
clockwise until it stops, then back to the
original position. This movement initiates
data collection at the detector.
23. The run will proceed until finished.
Chromatograms are saved
automatically. They can also be
annotated and printed at this point.
24. Run another equilibration for at least
five minutes to rinse the column.
25. When finished for the day, remove inlet
tubes from the mobile phase bottles
and close. Shut down pumps, column
oven, degasser and detector.
62
SOP HPLC-002
DIONEX HPLC WITH MANUAL INJECTION
Revision A, April 21, 2011
Title: Use and Maintenance of Dionex UltiMate 3000 HPLC with manual inject valve
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for use and maintenance of the Dionex UltiMate 3000
HPLC with manual inject valve.
2.0 SUMMARY OF METHOD
PART A: INTRODUCTION
The Dionex manual inject HPLC instrument
consists of several components:





an UltiMate 3000 Pump
a manual inject valve
an UltiMate 3000 Column
Compartment, consisting of a column
heater and a column
an UltiMate 3000 Variable Wavelength
Detector (VWD)
attached computer with Chromeleon 7
software
MATERIALS
1. Dionex UltiMate 3000 HPLC with
manual inject valve
2. Mobile phase solutions (HPLC
grade)
3. Sample for injection
4. Vacuum filtration assembly and 0.1
um nylon filters
5. 5 mL Luer-lok syringes, 0.2 um
cellulose acetate syringe filters
PART A: MOBILE PHASE PREPARATION
Prior to operating the HPLC, you must filter
and degas the mobile phase solutions.
Filter the solutions through a 0.1 um nylon
filter using a vacuum filtration assembly.
This filtration process will also degas the
solutions. Be aware that filtering 500 mL of
solution can take up to 30 minutes, so
provide ample time for this step or prepare
in advance.
Filter your sample for injection by passing
through a 0.2 um cellulose acetate syringe
filter.
PART B: OPERATION
63
Navigating in Chromeleon 7
The software will display a workstation that
consists of several tabs. Each tab
represents an HPLC system component
(pump, detector, etc), or a system function
(audit, startup, queue). The names mean:
TCC-3x000: column compartment
Ultimate 3000 Home: an overview of
the entire system
 VWD-3x000(RS): variable wavelength
detector
 xPG-3x000(RS,SD): pump
 Audit: displays an audit trail of every
action taken, all alerts the system issues,
and all errors that occur. This can be a
great way to detect the source of
failures.
 Startup: This is similar the the Home tab,
but it also displays the audit trail and
shows data from the detector and
temperature readings.This is a
comprehensive view of what's going
on.
 Queue: this tab will display all the runs
that have completed, as well as any
scheduled runs.
If you do not see these tabs displayed,
make sure that the Instruments category
bar is highlighted on the lower left side of
the screen.


Quick Guide
1. Power on HPLC components: pump,
column compartment, and detector
(switches are on back right of the
instrument and can easily be accessed
by reaching behind the right side of the
components). Power on attached
computer and double-click
Chromeleon icon.
2. If the Instruments category bar is not
highlighted blue at the lower left, click
it to enable. You must connect the
three instrument components by
toggling the Module Connect switch in
the software. Click on the column
compartment tab (TCC-3x000) and
located the Module Connect toggle
near the top left of the pane. Click on
the left side of the toggle to connect.
64
Give it a few seconds because the
toggle does not activate right away.
The green light will come on and the
module is now connected.
3. Repeat Step 2 with the pump (xPG3x00 tab). You must also start the pump
to begin equilibrating. Ensure that the
inlet tubing for the channels (A, B, C,
and/or D) you'll be using are
submerged in filtered, degassed
solutions of sufficient volume to purge,
equilibrate, and complete the
separations you plan to perform. The
flow rate should be set less than 1
mL/minute (0.6 mL/minute is a good
start). You can set this by typing in the
text box under the flow gauge (grey
rectangle to the right of the Module
Connect toggle). Then click the
Continue button (to the left of the
Pressure graph) to start the pump.
4. Once the pump has started, you need
to purge the pump (this is only
necessary when you start the pump at
the beginning of the day or when you
change eluents). Click the check box
next to the box that says "Purge:Off".
You will get a warning stating that you
should open the purge valve screw. Lift
the pump faceplate on the front of the
pump to expose the purge valve. The
valve screw is brass and in the center of
the assembly. Turn counter clockwise
about a half turn (if you open too much
it will leak). Then click the button on the
alert dialog box that says "Execute
despite warning". The pump will
increase the flow to 3 mL/minute and
flush air bubbles out as well as clear
any old eluent from the system.
Continue until no air bubbles are
coming out of the waste line that
comes directly from the purge valve.
Click the check box next to Purge to
slow the pump and then tighten the
valve screw (do not tighten the screw
first or you could damage the
column!).
5. Repeat step 2 with the detector tab
65
(VWD-3x000). You must also ignite the
lamp(s) you plan to use; turn on the
lamp(s) by clicking the check box next
to the name, UV and/or Vis.
6. Once all the components are
activated and ready to use, you need
to create a sequence. A sequence is a
program for the chromatography run(s)
you would like to perform in a given
session. This can consist of one or more
injections using one or more instrument
methods. An instrument method is the
program for how you would like the
instrument components to behave
during a chromatography run.
Instrument methods can be used again
and again, while a sequence can only
be run through once and then is saved
as a data file. If you have not yet
created the instrument method you
would like to use for your sequence,
skip to step 8 and come back to 7
when you have created a sequence.
7. Create a sequence by clicking Create
on the menu bar at the top of the
screen. This will start the Sequence
Wizard which will help you create a
sequence. You will select the number
of injections (this can be increased
later if desired), the instrument method
to be used (if you add injections later
you can also use a different method if
desired), and then save the sequence
with a specific name.
8. Create an instrument method by
clicking on the down arrow next to
Create on the menu bar at the top of
the screen and clicking on Instrument
Method. This will start the Method
Wizard which will help you create a
method. You will select which
instrument components to use (the only
optional one is the column
compartment which controls the
temperature of the eluent), and
program their parameters. For
example, you will program the pumps
for the flow rate and eluent
composition and the length of time
66
they spend pumping at those settings.
9. When you have the sequence and
method(s) how you want them, you
are ready to begin a run. Insert a flat
tipped syringe containing your sample
into the manual inject valve. You
should have at least three times the
volume of the sample loop in the
syringe (standard loop is 20 uL). Ensure
that the inlet tubing for your eluent(s)
are submerged in the liquid and that
you have sufficient volume to
complete your run(s). The pump should
also be running at the same flow rate
as your starting rate for the method
you're using (this ensures a smoother
baseline). Check the Queue tab for
leftover sequences and clear them by
clicking remove (on the right of the
screen). If you do not do this, the
software will run through those before
the one you want to do.
10. Open the sequence (if it isn't already
active) by activating the Data
category bar on the bottom left of the
screen and locating your sequence in
the menu tree and clicking it. Click the
Start button at the top of the pane to
begin. The software will perform a
Ready Check to ensure that all
components are ready to be used; at
this point it is common to encounter
errors or alerts. Usually errors are
accompanied by an indication of how
to correct the error. Alerts may require
no action but should be noted. See
troubleshooting section for a
description of common errors and
alterts (COMING SOON!).
11. Once the Ready Check is complete
you won't see a prompt asking you to
inject, but you should inject now
(except if there are any errors that
need fixing). Load the sample loop by
pressing the syringe plunger to release
at least three times the volume of the
loop. Rotate the valve lever by pressing
down on the black plastic lever firmly
and leave it in the inject (down)
67
position for about 30 seconds (this time
might be longer if the loop is
significantly larger, but this is not
common). This gives the system time to
inject the contents of the loop onto the
column. After 30 seconds return to the
load (up) position.
12. When you pressed the lever down, the
system detected this movement and
initated data acquisition at the
detector. If you go to the Instrument
category bar and go to the detector,
home, or startup tabs, you will see a
graph with data traces just starting to
appear. You can now leave the
instrument if needed and it will
continue automatically until the current
injection has completed its method. If
you programmed more than one
injection, you will need to press start
again and perform the next injection;
however, the system will stay on hold
between the injections so you don't
need to be at the instrument right
when the previous segment ends.
13. When a segment of the sequence is
complete, you may begin data
processing. Double-click the
chromatogram thumbnail to the left of
the name of the injection to open the
data file. A new window opens
containing a data processing browser
(called the Chromatography Studio).
You will see the chromatogram of the
selected wavelength trace(s). Below
the chromatogram, you can see an
empty table. Peak information will
appear here when you have chosen a
processing method. A processing
method is a program telling the
software how to recognize peaks and
integrate the area under them. In the
panel above the chromatogram you
will see a button that says Processing
Method. Click it and this will move the
table down to reveal an Open File
icon. Double click this and you will be
able to choose from an existing
processing method. If none of the
existing methods are what you want,
68
go back to the Chromeleon console
and use the Processing Method Wizard
to create a new method, then go back
to the Chromatography Studio and
choose it.
14. When you have done your data
processing, you may obtain a copy of
your data; press the Print Screen key on
the keyboard, and then paste this
image into Microsoft Paint. You can
then save this file as a BMP or JPEG and
save to your flash drive.
15. When finished for the day, stop the flow
of the pumps by clicking the Stop Flow
button in the pump tab, turn off the
lamp(s) by unchecking the box next to
them, and then power off all three
components.
69
SOP INC-001
HERAcell CO2 Incubator
Revision A, April 21, 2011
Title: Use and Maintenance of HERAcell 150 CO2 Incubator
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for use and maintenance of the HERAcell 150 CO2
incubator.
2.0 INTRODUCTION
The HERAcell 150 CO2 incubator is a laboratory device for preparing and culturing cell and tissue
cultures. The device allows the simulation of the special physiological ambient conditions for
these cultures due to the exact control of temperature, CO2 and O2 content, and increased
relative humidity. In the ACC Biotech program, this incubator is intended to be used to grow
cultured animal cells.
3.0 SUMMARY OF METHOD
MATERIALS



HERAcell 150 CO2 incubator
50 lb tank of medical grade CO2 with
regulator to deliver 0.8-1 bar
Sterile tap water, 3 L
PART A: SET UP
These instructions are for starting the
incubator up when it has been off for a
while.
1. With the incubator turned off, open the
exterior and interior doors. The interior
door can be opened by rotating the
blue handle.
2. Inspect the interior of the incubator for
cleanliness; if it needs cleaning, use a
paper towel and 70% ethanol. Allow
the ethanol to evaporate before
turning on the incubator.
3. Pour 3 L of sterile tap water into the
reservoir at the bottom of the interior of
the incubator.
4. Close the doors. Make sure the
Medical grade CO2 can be ordered from
Praxair.
70
incubator is connected to a 50lb tank
of medical grade CO2 with a regulator
that can deliver 0.8-1 bar (10-20 psi).
5. Open the valve on top of the gas tank
(rotate about 3 full turns). Then open
the black plastic valve on the side of
the regulator (rotate about 3 full turns).
Both gauges on the regulator should
indicate pressure. Do not move the
white valve yet.
6. Turn on the incubator by pressing the
on/off switch on the bottom front. Wait
a few moments for the incubator to
initialize and display the current
temperature and CO2. These will most
likely be below the set points of 37ºC
and 5% CO2, so the yellow indicator
lights will be on.
7. Go back to the gas regulator. Look at
the gauge on the left; it should register
approximately 10 psi. If it is significantly
above or below this value, CAREFULLY
AND SLOWLY adjust the white valve
until the pressure is between 9 and 11
psi.
8. Allow the incubator to equilibrate to
the set point temperature and pressure.
The relative humidity inside the
incubator will increase as the water
from the reservoir evaporates into the
warm air. No need to change any
settings for humidity.
9.
After 10 minutes check the
temperature and % CO2. If the
temperature is too low, give the
incubator some more time to
equilibrate. Confirm the temperature
using a certified thermometer or
temperature probe.
10. If the CO2 is too high or low, check the
regulator gauge. The right gauge
should display several thousand PSI
while there is gas in the tank (this
cannot be adjusted). The left gauge
should be user-adjusted to around 10
psi.
IMPORTANT: the CO2 inside the tank is
under such high pressure that some of it is
liquefied. As gas is consumed and leaves
the tank, the liquid amount grows smaller;
however, the same pressure will register on
the regulator as long as there is liquid CO2
in the tank. What this means is that you will
71
11. When the temperature and % CO2
have reached the set points, the
humidity will also have equilibrated to
the appropriate level and cells can be
safely placed in the incubator.
12. Only open the interior door when you
need to add or remove items from the
incubator. Try to coordinate these
openings with other users to minimize
the total number of openings per day.
When you open the interior door
quickly remove or replace your item
and close the door; do not leave the
interior door open for any reason if the
incubator is on.
PART B: MAINTENANCE
1. Be sure to record every opening and
closing of the incubator in the
equipment log. This helps indicate
when a new gas tank will be needed.
2. Check the water level in the water
reservoir occasionally. It should come
within a few inches of the mark in the
bottom of the incubator. If it seems low,
add sterile tap water 500 mL at a time.
Do not overfill!
3. No regular maintenance of the
incubator is required. However, when
the incubator will not be used for
longer than a week, shut it down to
save CO2. Turn the incubator off. Close
the main valve on the top of the gas
tank and then close the black side
valve. If the incubator will not be used
for more than a month, remove the
water in the reservoir using a vacuum
setup and dry the reservoir with paper
towels.
not observe a decrease in the pressure of
gas in the tank UNTIL there is almost none
left, then the pressure will rapidly decrease
(within the same day or so). Therefore, it is
important that the tank is monitored and a
spare tank is kept on-site so they can be
switched out quickly when the original tank
empties.
72
SOP MCP-001
USE OF VWR MICROPIPETTORS P10, P20, P100, P200 AND P1000
Revision A, August 15, 2012
Title: Operation and maintenance of VWR Micropipettors
1.0 SCOPE AND APPLICATION:
This SOP outlines the procedure for operation and maintenance of VWR P10, P20, P100, P200 and
P1000 micropipettors.
2.0 SUMMARY OF METHOD
PART A: OPERATION
COMMENTS
1. Each student or group should check
out a set of micropipettors for the
duration of the course. Your instructor
will tell you how to record your
selection and if you need to validate
your set (see Part C). If any
micropipettors in your set are
damaged, notify your instructor
immediately.
Check-out will prevent abuse of
micropipettors, and ensure that errors in
measurement can be tracked to an out-ofcalibration instrument.
Note that the most common measuring
range for a P10 is 1.0-10.0µL, for P20, 2.020.0 µL, for P100, 10.0-100.0 µL, for P200,
20.0-200.0 µL, for P1000, 100.0-1000.0 µL.
Never adjust a micropipettor outside of this
range!
On P10, P20, P100, and P200
micropipettors, the black digits indicate
microliters; the red digits indicate tenths
and hundredths of microliters. On P1000
micropipettors, the red digits indicates
milliliters and the black digits indicate
microliters.
The appearance of the minimum and
maximum volumes on the analog readout
of each size micropipettor are shown in the
appendix to this SOP.
2. Select a micropipettor from your set
that will measure the volume you need.
3. Set volume using volume adjustment
knob. Hold micropipettor in one hand,
and with other hand, turn volume
adjustment knob counterclockwise so
volume indicator is 1/3 revolution
above desired setting, then turn slowly
clockwise until indicator shows desired
volume. If micropipettor dial is past
either its high or low limits, or the dial will
not rotate, notify your instructor
immediately.
4. If you pass the desired setting, turn dial
1/3 revolution higher than desired and
reset volume.
5. Attach a new disposable tip to the
pipette shaft by pressing shaft into tip
while tip is still in box. Press only hard
enough to make a positive airtight seal.
6. Press plunger to first stop. This part of
the stroke is volume indicator.
Always dial down to the volume setting.
This prevents mechanical back lash from
affecting accuracy. Otherwise, precision
and accuracy will be affected.
Do not touch the tips. To avoid
contamination a fresh tip should be used
for each measurement.
Never immerse the tip more than a few
millimeters below the surface of the fluid. A
deeper immersion can cause fluid to cling
to outside of tip, delivering more fluid than
was desired.
73
7. Holding micropipettor vertically
(perpendicular to the bench), immerse
the last few millimeters of the
disposable tip in the fluid to be
measured.
Never let the plunger snap up. This can
cause air bubbles to fill the tip, destroying
accuracy.
8. Release the plunger slowly until it has
returned to its original position.
9. Pause for a few seconds to ensure that
the full volume of fluid is drawn into the
tip.
10. Withdraw the tip from the sample,
keeping the micropipettor completely
vertical.
Always keep the micropipettor in vertical
position to ensure that no liquids enter the
micropipetter or drip from the disposable
tip.
11. Inspect tip to make sure there is no air
bubble on the inside and there is no
solution on the outside of the tip.
12. If there is an air bubble inside tip, you
must pipet this volume back into the
sample and measure again, repeating
steps 5-10.
13. If there is solution on the outside of the
tip, touch tip to the wall of the solution
container to remove it.
14. To dispense the sample touch the tip
end against the side wall of the
receiving vessel and depress the
plunger slowly to the first stop, then
press the plunger to the second stop,
expelling any residual liquid in the tip.
Dispense the fluid into the lowest possible
place in the receiving vessel.
15. With the plunger fully pressed, withdraw
pipette from the vessel carefully, with
the tip remaining against the wall of
the vessel.
16. Release the plunger to return to the up
position.
17. Discard the tip into a waste beaker or
trash receptacle by depressing the tip
ejector button.
Never discard tips into sinks. Always use a
waste beaker or trash receptacle.
If you measure a hazardous material
(chemical or biological), dispose of
contaminated tips into a properly labeled
74
PART B: TIP SELECTION
1. Tips must seal properly on the shaft to
assure an airtight seal and avoid leaks
or poor accuracy.
2. Tips must be soft and flexible so that the
shaft is not scratched or worn
prematurely.
container and store with the hazardous
waste.
Barrier tips provide the best protection from
air contamination, but they do not always
fit on the smallest micropipettors.
Do not use sterile tips for non-sterile
applications. Do not use gel loading tips for
non-gel-loading applications. These tips
are expensive.
3. Tips must be free from microscopic
particles.
4. The tip orifice must be the correct size,
and orifice size and geometry must be
consistent from tip to tip.
5. Interior and exterior surfaces must be
clear, smooth and hydrophobic to
avoid retention of liquid.
PART C: VALIDATION
1. Micropipettes should be validated at
least once a semester.
2. Obtain a printed copy of the blank
Micropipettor Validation Log
(Appendix). Record the serial numbers
and set number of your micropipettor
set. Also include the names of people
in your group.
3. Locate an analytical balance and
calibrate if necessary.
4. Dial a micropipettor to its highest
volume.
5. Place a weigh boat on the weighing
pan of the balance, close the draft
shield, and tare the balance.
6. When balance is stable, measure
highest possible volume of purified
water with the micropipettor, and
dispense into the weigh boat in the
balance.
7. Close the draft shield and wait for the
balance to stabilize. Record the mass
Your instructor will tell you if this process has
been assigned to your class.
75
of the water in the validation log.
8. Repeat steps 6 and 7, adding each
measurement of water to the weigh
boat and recording the total mass of
water with each addition.
9. Make ten measurements. Discard the
weight boat with water.
10. Repeat steps 4 through 9 with each of
the other micropipettors in your set.
11. When complete, enter the data into
the Excel file, Micropipettor Validation
Log Template.
12. Save and print or email the file to your
instructor.
Functions programmed in the workbook will
automatically calculate the volume
delivered in each measurement, and the
resulting percent precision.
76
APPENDIX TO SOP MCP-001
USE OF VWR MICROPIPETTORS P10, P20, P100, P200 AND P1000
Updated May 19, 2006
Appearance of Analog Readout at Minimum and Maximum Volumes of Micropipettors
P1000 (100-1000 μL)
Minimum, 100 μL
0
1
0
Maximum, 1000 μL
0
2
0
Maximum, 1000 μL
0
2
0
Maximum, 200 μL
0
5
0
Maximum, 200 μL
1
0
0
P1000 (200-1000 μL)
Minimum, 200 μL
1
0
0
P200 (20-200 μL)
Minimum, 20 μL
2
0
0
P200 (50-200 μL)
Minimum, 50 μL
2
0
0
P20 (2-20 μL)
Minimum, 2 μL
0
2
0
Maximum, 20 μL
2
0
0
P10 (1-10 μL)
Minimum, 1 μL
0
1
0
Maximum, 10 μL
1
0
0
77
SOP MIC-001
NIKON TS100 INVERTED MICROSCOPE
Revision A, May 27, 2011
Title: Use and Maintenance of Nikon TS100 Inverted Microscope
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for use and maintenance of the Nikon TS100 Inverted
Cell Culture Microscope.
2.0 SUMMARY OF METHOD
MATERIALS



Nikon TS100 inverted microscope
Flask or slide with cells
Lens papers
PART A: INTRODUCTION
The Nikon TS100 inverted microscope is
intended mainly for use in microscopic
observation and in the micromanipulation
of living cells and tissue using transmitted
and reflected illumination.
Our RRC cell culture lab is equipped with
six TS100 microscopes. These microscopes
have the light source and condenser on
top and the objectives on the bottom,
allowing enough space on the stage for
cell culture plates and flasks. Brightfield
illumination is provided by an incandescent
bulb. Each condenser contains a phase
slider that results in adjustment-free phase
contrast images. The TS100 model is very
durable and can stay functional as long as
20 years with proper care.
PART B: OPERATION—PHASE CONTRAST
1. Turn on the lamp using the green on/off
switch at the bottom left. Adjust the
brightness of the viewfield with the
brightness adjuster (rheostat).
2. Adjust the interpupillary distance.
Looking through the eyepieces, adjust
the distance between the binocular
eyepieces so that the left and right
viewfields overlap to form a single
The condenser is located just below the
lamp housing. There are two inserts in the
condenser. The top insert is a colored glass
filter; the default is a blue filter that reduces
eye strain compared to the unfiltered
incandescent light. There are other color
filters available that might be used in
special situations.
78
image.
3. Set the diopter adjustment ring to the
reference position. Turn the ring on
each eyepiece to align the 0 line with
the reference line.
4. Fully open the aperture diaphragm.
Move the aperture diaphragm level on
the condenser to the full right to fully
open the aperture.
5. Set the specimen in place. Place a
flask, dish, or slide on the stage. Adjust
its position so that the center of the
container comes under the optical
path. Be careful that the ring insert in
the stage is compatible with the vessel
you are viewing (i.e. it will not fall
through the stage).
Before placing large containers on the
stage, be sure that the tip of the objective
does not protrude past the stage or it may
be damaged.
6. Adjust the diopter. If necessary, adjust
the diopter adjustment rings according
to the power of your left and right eyes.
7. Observe the specimen. Start with the
lowest objective and focus using the
coarse adjustment knob, being careful
not to hit the specimen with the
objective.
8.
When focused, rotate the nosepiece
to a higher power objective if desired.
Focus again, using the fine adjustment
knob. Adjust illumination using the
rheostat if desired.
9. When finished, lower the nosepiece
with the coarse adjustment knob and
rotate to the lowest objective. Turn off
the microscope and cover.
PART C: MAINTENANCE
Because the TS100 is equipped with a
phase contrast slider, the Kohler
illumination does not need to be checked
or aligned. In fact, aside from the individual
adjustments of oculars and condenser, this
microscope requires virtually no
adjustments.
DO NOT rotate the left and right focus
knobs in opposite directions at the same
time or continue to rotate the coarse focus
past the upper or lower limit of its motion.
These actions will damage the microscope.
*When rotating the nosepiece, grip by the
knob and not by the objective because
they can become damaged by twisting or
can come loose.
79
Illumination is emitted from the bulb in the
lamp housing (black cylinder at very top).
The bulb will provide light until it burns out.
To change the bulb, squeeze the lamp
housing and pull up. Pull out the bulb and
insert a new one (handle only with gloves—
oils on your hands will shorten the life).
One a year, the microscopes should be
cleaned professionally. We have a oneyear parts and labor warranty with Nikon,
as well as a 5-year parts warranty (for
everything but electronics).
PART D: TROUBLESHOOTING
If there is no illumination, make sure the
microscope is switched on, plugged into
the wall, and that the power cord is firmly
connected to the device (sometimes the
cords can come loose). Try turning the
rheostat completely up or down. If no
illumination occurs, the lamp needs to be
reinstalled or replaced.
If the viewfield is only partially visible, the
nosepiece may not have clicked into
place (the objective is not in the optical
path). Rotate until it stops with a click.
There is a torque adjustment wheel on the
coarse/fine adjustment knob that can be
used to tighten or loosen the coarse and
fine adjustment. If this is too loose, the
stage can slide down with gravity. Try to
keep the torque at a moderate level and
do not over-adjust it.
80
SOP MIC-002
NIKON 55i FLUORESCENCE MICROSCOPE
Revision A, April 21, 2011
Title: Use and Maintenance of Nikon Fluorescence Microscope
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for use and maintenance of the Nikon Eclipse 55i
Microscope with Fluorescence Accessory.
2.0 SUMMARY OF METHOD
MATERIALS






Nikon 55i upright light microscope
Fluorescence light source and power
supply
Camera and control center/view
screen
Fluorescent sample on slide for viewing
Immersion oil (if using 100x)
Lens papers
PART A: INTRODUCTION
The Nikon fluorescence microscope can
be used to observe fluorescently labeled
cells or intracellular structures. It consists of
a basic Nikon 55i upright light microscope,
fluorescent light source and power supply,
CCD camera, and a camera control
center/view screen. The CCD camera
takes high-resolution images and displays
them on a view screen that is also the
camera control center. The control center
has firmware that can be used to capture,
save and annotate the images.
There are 20x, 40x, and 100x objectives
installed; oil immersion can be used at the
100x power.
PART B: OPERATION
Fluorescence Illumination
1. Turn on the fluorescence power supply
using the toggle switch. Turn on the
microscope and camera control
center.
*It is not necessary to warm up the metal
halide lamp before capturing images (this
only matters if you are quantitating
fluorescence intensity and we cannot do
that with the software we have).
When using fluorescence, turn the power
supply on before the microscope and
camera control, because the surge of
power from the metal halide lamp could
possible damage the optics of the
camera. For the same reason, turn the
power supply off last. After the metal
halide lamp has been turned off, there is a
20-minute auto delay before it can be
turned back on. If you try to turn it on
81
2. Turn off Brightfield illumination by
turning the rheostat down until no light
shines through the condenser. The
Brightfield condenser must be turned
off in order to see fluorescent images.
Additionally, you should also cover the
condenser with something dark and
non-reflective (like dark matte fabric or
paper) because reflected ambient
light can also saturate the pixels of the
camera and drown your fluorescent
image out. You should also turn off
overhead lights, especially fluorescent
lights, because they will affect the
quality of the image you see.
3. Select the filter cube appropriate for
the fluorophore you are looking at in
your sample.
We currently have three filter cubes: for
DAPI, FITC, and TRITC stains. The FITC will
also work to visualize GFP. The DAPI can be
used for Hoescht mycoplasma stain. These
are all long-pass filters, which will allow you
to visualize any fluorescence at a
wavelength above the emission filter
wavelength. So, you may see other colors
of fluorescence besides the one you are
looking for. There are also band-pass filters,
which allow just a narrow range of
wavelength light through. These are
necessary when quantitating fluorescence
intensity because you should only count
emission of a single color.
Filter positions:
1
empty
2
DAPI (UV)
3
FITC (GFP)
4
Rhodamine (TRITC)
4. Place your sample slide on the stage
between the clips until square. Rotate
the nosepiece until the objective you
want to use is over the sample (should
be 20X or 40X at this point). You should
see some type of image on the view
screen. You do not have to use the
oculars to view the sample, as the
image should appear in the view
screen just as it does in the oculars (with
the exception that the oculars can be
during the delay, the light will flicker.
Make sure to always turn off the light
source when not in use.
The rheostat is a small knob located on the
right side of the microscope body.
*The light passes through one of several
filter cubes, which will only allow one
wavelength of light through to the sample.
This excites molecules in the sample, which
emit a higher wavelength light; this light
passes through another filter on the cube
and then travels to the camera and your
eye. The filter cubes rotate around in the
nosepiece via a knob on the right side. The
number on top of the knob corresponds to
the active filter. There is also a filter lock
toggle switch on the front of the
nosepiece. When activated, you cannot
switch the filters. Keep this out at all times
so you can switch the filters.
*When rotating the nosepiece, grip by the
knob and not by the objective because
they can become damaged by twisting or
can come loose.
The oculars (eyepieces) are individually
adjustable and also telescope out and
82
adjusted for individual eye differences).
5. If the image appears too bright on the
view screen, you can engage one or
more of the neutral density filters to dim
it.
6. Using the coarse adjustment knob,
adjust the stage up or down to focus
on the sample until you achieve a
good image. Then use the fine
adjustment knob to fine-tune the focus.
If desired, rotate the nosepiece to
select a higher magnification and fine
adjust again.
7. When the image you have on the view
screen is satisfactory, you may capture
images
Capturing Images
The CCD camera will capture what you
see in the ocular and display it on the view
screen/control center.
1. To capture an image, simply press
the Capture button on the view
screen.
Under the CAM menu there are many
settings that can be changed to obtain a
better image, including contrast, color,
exposure time, etc. There are some presets
that are comparable to scene settings on
your digital camera. You can also save
your own settings and recall them later.
The control center has firmware that can
be used to annotate and customize
images. There are also annotation tools
that can be used to draw lines, circles,
labels, and other features, and you can
also calculate distances between
structures or the area of a region. These
can be found under the TOOL menu.
Use a USB storage device to save your
images. You can insert the device directly
into the control center. There is also an 8GB
memory card that can be used if you
forget to bring a USB storage device. The
files are saved with the file name stem
down for ergonomics.
*There are a series of neutral density (ND)
filters on the microscope body and on the
lamp power supply. These will dim the
intensity of the light without changing the
color. This can be helpful to prevent
photobleaching of a sample from overintense light. The number on the filter (2, 4,
8, 16) corresponds to the factor by which
the light is reduced (by half, ¼, etc).
*There is a torque adjustment wheel on the
coarse/fine adjustment knob that can be
used to tighten or loosen the coarse and
fine adjustment. If this is too loose, the
stage can slide down with gravity. Try to
keep the torque at a moderate level and
do not over-adjust it.
* There is a knob on the front of the
microscope that opens and closes the
shutter for fluorescence illumination. You
can close this if you want to stop
illuminating temporarily, but do not want to
turn off the lamp (because of the 20minute delay). You might want to use this if
you have to walk away from your
microscope for a few minutes, but want to
avoid photobleaching the sample on your
slide.
83
“IMGBOX” and then with the time and
date. You can change the file name stem
but the time and date will still be included.
This menu can be reached from the SETUP
menu on the main screen.
You will have the option to save files as
JPEG, BMP, or TIF. These files are the same
format as digital camera files so they
should open with no problem on virtually
any operating system. There is no attached
printer, so you will need to print on a
regular office printer.
The VIEW menu allows you to recall saved
images.
Under the REC menu, you can command
the firmware to take a series of timelapse
photos (not at video speed, but similar to a
video).
PART C: MAINTENANCE
Brightfield illumination is provided by an
LED light that produces very little heat and
never needs replacing. LED light also
causes less eye strain than traditional
incandescent light. The condenser does
not have phase contrast.
The fluorescent lamp is housed inside the
power supply. It is a metal halide lamp that
lasts about 2,000 hours. There is a counter
on the power supply that records the hours
that you can reset when you replace the
bulb; it should last around 2000 hours. A
replacement lamp costs $695 and it is very
easy to replace, but a technician from
Nikon can do this if you buy the lamp.
PART D: TROUBLESHOOTING
1. After the metal halide lamp has been
turned off, there is a 20-minute auto
delay before it can be turned back on.
If you try to turn it on during the delay,
the light will flicker.
2. On the bottom right of the microscope
84
body there are two rods that are used
as an on-off switch for the color
balance filter. The top rod in is the “on”
position. Activate this if you perceive
the images as being too blue when
using Brightfield, which can happen
because of the LED illumination.
3. On the bottom right side of the
microscope body there is a small white
button that says PRE (for “preset”). This
is the autophoto button, which will
freeze all light settings. This can be
helpful if you want to take several
images with the same settings and
don’t want to accidentally change
anything. If the light controls will not
change even when you move the
controls, make sure this button is not
pressed in.
4. The light from the lamp travels from the
power supply through a thick fiber
cable to the microscope body. It is very
important to not bend or crush this fiber
because it will dim the intensity of your
illumination. If you are having
illumination problems, one of the best
quick fixes is to replace this cable.
5. If there is a discrepancy between the
images you see in the ocular and on
the view screen, first adjust the ocular
to account for your eyesight. If they still
don’t agree, you can adjust the
parfocal adjustment control which is a
wheel under the camera.
6. The camera on/off slider is located on
the top right of the microscope body.
When in the on position, this causes the
image to be split between the
eyepieces and the camera. It must be
on when taking photos, but need not
be on when simply observing
specimens. If your signal is dim while
observing, you can try turning this off
until you need to take a photo.
85
SOP MPR-002
USE AND MAINTENANCE OF BIOTEK SYNERGY 4 MICROPLATE READER
Revision A, June 8, 2011
Title: Use and Maintenance of Synergy 4 Microplate Reader
1.0 SCOPE AND APPLICATION:
This SOP outlines the procedure for use and maintenance of the BioTek Synergy 4 Multimode
Microplate Reader.
2.0 INTRODUCTION
The Synergy 4 multimode microplate reader is capable of UV and visible absorbance readings,
as well as bottom- and top-reading fluorescence. It can only be operated using the Gen 5
software on an attached computer. The Gen 5 software can be installed on up to five
computers. Computers must have Windows XP or Vista, Microsoft Excel for exporting data, and
at least 1 GB of RAM.
The reader contains both a tungsten lamp with a filter wheel and a xenon flash lamp with a
monochromator. Continuous wavelength tuning using the monochromator is available for both
absorbance and fluorescence modes. There is a limited selection of filters provided; however,
additional filters can be ordered. The advantage of filters is increased sensitivity and specificity.
The tungsten lamp requires 3 minutes to warm up. The xenon flash lamp does not require a
warm-up period.
The reader can be programmed to incubate at temperatures from ambient to 42ºC. The
incubator requires 15 minutes to warm up. It can also be programmed to shake plates before
reading.
In absorbance mode, the reader can measure up to 6 wavelengths at a time. It can also obtain
an absorbance spectrum of each well.
Greiner brand 96-well plates are recommended. For UV absorbance, use clear plates coated
with a substance to make them transparent to UV. For visible absorbance, any transparent
plastic will work. For top-reading fluorescence, use solid black plates; for bottom-reading
fluorescence, use solid black plates with clear bottoms. Never use lids on plates because they
will make the plate too tall and the lid will damage the optics. Avoid using sealing film or tape
on plates because the optics will be located very close to the top of the plate.
3.0 SUMMARY OF METHOD
COMMENTS
PART A: OPERATION USING QUICK READ
1. Turn on lamp power supply (gold box
connected to power cord) and
instrument (switch is on front panel). The
instrument will initialize; some noises are
normal.
2. Connect laptop computer to the
instrument via a USB cable and start
computer. Open the Gen 5 software.
3. At the Welcome screen, click “Read a
Plate.”
86
4. Design a method by adding steps
(such as Read, Shake, Delay,
Temperature, etc). You must add at
least one Read step, but the other
steps are optional.
5. When you add a Read step, you will be
given the following options:
a. Detection Method (choose
Absorbance)
b. Read Type (choose Endpoint)
c. Read Speed (choose Normal)
6. Choose the number of wavelengths
and enter them in the drop-down
menu(s). Click OK when finished with
this Read step.
7. Enter Delay, Temperature Set, Shake or
more Read steps if desired. When you
have finished designing your
procedure, you may click Validate to
make sure the order of your steps is
logical (this is only useful when you
have entered more than one step).
When designing a protocol, you will be
able to choose your plate manufacturer
from a drop-down menu. The design of
each plate will affect the protocol slightly
(usually, the plate height affects the
distance of the optics from the plate).
Therefore, it is important to choose the
correct plate from this list. If it is not on the
list, choose Generic.
If you have full wells (300 uL), do not use
the fast shake option because your sample
may splash out of the plate. Plates should
shake for 3-5 seconds, followed by a 2second delay before reading.
If you do not turn on the Tungsten lamp,
the Xenon flash lamp will be used. The
Xenon lamp does not need to warm up
before use.
8. Click OK when finished designing your
procedure. Enter a plate ID on the next
menu and add comments if desired.
9. Click Read. The Save As dialog box will
open; give your data set a unique
name and click Save.
10. The sample tray will open. Insert your
plate with well A1 oriented towards the
upper left corner. Click OK, the tray will
close, and the reader will scan the
plate.
11. The tray will open when the procedure
is finished. Remove the plate and
manually close the tray by pressing the
black eject button on the front panel of
the instrument.
12. A window with a schematic of the
plate containing data will appear. You
may need to choose the wavelength in
the Data drop-down menu to see the
data.
No more than 350 µL should ever be
added to a microplate for use in this
instrument, due to the risk of spills.
87
13. To export data to Excel, click the Excel
icon next to the Data drop-down
menu.
14. To read another plate with the same
procedure, click the Add Plate icon on
the toolbar. Follow the above
directions, starting with entering a plate
ID.
PART B: MAINTENANCE
1. Routine maintenance of the instrument
should include cleaning the plate
carrier to remove sample residue (an
alcohol prep pad can be used) and
blowing dust from the filters using
canned air. This should be performed
about once every six months.
2. To keep the reader in optimal
condition, keep the plate carrier clean
and free from spills, dust, and debris.
Never leave plates in the reader after
reading them, because water will
evaporate and condense inside the
instrument, ruining the optics.
3. About once a month, run a System Test
to confirm that the reader is working
properly. Choose System > Diagnostics
> Run System Test. The reader will run a
series of diagnostics to evaluate
performance and will pass or fail. If the
reader fails the test, contact Technical
Support with the error code provided.
Most failures are lamp-related.
88
SOP PCR-001
MyiQ iCycler Real-time PCR System
Revision A, June 8, 2011
Title: Use and Maintenance of MyiQ iCycler Real-time PCR System
1.0 SCOPE AND APPLICATION:
This SOP outlines the procedure for use and maintenance of the iCycler thermal cycler with the
MyiQ Single-color Real-time PCR Detection System.
2.0 SUMMARY OF METHOD
MATERIALS


iCycler with MyiQ optical module
Computer with iQ5 software
PART A: SETUP
1. Check that the base unit (thermal
cycler) is connected to a USB port on
the computer. The base unit should
also be connected to the optical
module (upper unit) by a serial cable.
Both units should be plugged in to
power outlets.
2. Turn on the base unit using its power
switch; turn on the MyiQ optical
module using its power switch.
3. Double-click the Bio-Rad iQ5 icon on
the computer desktop.
4. Perform background calibrations (see
Appendix 1).
5. Choose the Workshop tab; highlight the
Setup tab, then the Protocol tab.
6. To select an existing protocol, click the
protocol name. The selected protocol
appears in the Selected Protocol
window.
7. Edit a selected protocol by clicking Edit
in the Selected Protocol window.
8. Create a protocol from a protocol
template by clicking Create New in the
Selected Protocol window.
89
9. Edit the protocol by performing one or
more of the following five tasks:
a. Edit the Dwell Time and Setpoint
temperature. Click in the Dwell Time
or Setpoint cell, and then enter the
Dwell Time or Setpoint temperature.
To enter 10 seconds, type 0
followed by : then 10 (that is, as the
time appears in the spreadsheet).
Alternatively, 10 seconds can be
entered as 0.10.
b. Acquire data. Click in the Data
Acquisition column, and then click
Real-Time at the step you want to
collect real-time data. Click Melt
Curve if data from a melt curve is
required.
c. Insert cycles and steps. Insert a
cycle by clicking in the Insert
column within the cycle row. Cycles
have a blue background. iQ5
software inserts the new cycle
below the current cycle. Insert a
step by clicking in the Insert column
within a step row. Steps have a
white background. iQ5 software
inserts the new step below the
current cycle.
d. Delete cycles and steps. Delete a
cycle by clicking in the Delete
column within a cycle row. Cycles
are indicated with a blue
background. Delete a step by
clicking in the Delete column within
a step row. Steps are indicated with
a white background.
e. Save the protocol. Click Save & Exit
Protocol Editing. Type the name of
the protocol in the Save As dialog
box, and then click Save.
10. Create a plate setup by clicking the
Create New button under “Selected
Plate setup.”
11. Enter a file name, and enter any notes
about the plate setup in the Notes box.
12. Enter or edit the sample volume, seal
type, and vessel type.
13. Enter or edit a name for the
experiment.
90
14. Click a sample type icon.
15. Select the type of replicate loading
desired.
16. Click on FAM under “Fluorophore.”
17. Click or drag across the plate to define
wells with the selected fluorophore
(FAM) and sample type.
18. Continue defining the remaining wells
by changing to the other sample type
icons.
19. To delete a previously defined well,
click the Erase Well(s) icon and then
click the well.
20. To delete the selected fluorophore
from a previously defined well, click the
Erase Well(s) by Selected Fluorophore
icon and then click the well.
21. If there are standards in the first dye
layer, click Dilution Series and calculate
the concentrations of the standards, or
type the concentrations in the
spreadsheet manually.
22. Click Save & Exit Plate Setup. Give the
plate setup file a new name or
overwrite the current version of the file.
The software adds the extension .pts
automatically.
PART B: OPERATION
1. In the Protocol tab, click Run. The
Initiate Run screen will appear.
2. Select Use Persistent Well Factors.
3. Click Begin Run.
4. Name the file in the Save Optical Data
File dialog box and click OK.
5. Run will initiate and the Monitor Run
window will open.
6. Thermal cycler will hold amplicons at
4ºC overnight if necessary.
91
PART C: TROUBLESHOOTING
PROBLEM:
 After opening the software, an error
message appears: “Cannot detect
camera or thermal cycler.”
SOLUTION:
 Check that the serial cable is
connected between the base unit
and optical module. Check that
the base unit is connected to a USB
port on the computer. You may
have opened the software before
the base unit finished initialization.
Turn off base unit and optical
module, close the software, turn
base unit and optical module on
and allow them to initialize. Then
open the softare.
92
Appendix 1: Calibration of MyiQ Detection System
NOTE: The detection system needs to be calibrated once a month, or whenever users change
sample volume or vessel type. Therefore, this procedure should generally be performed each
time the detection system is used.
MATERIALS
1. iCycler with MyiQ detection system
2. External Well Factor Solution (Bio-Rad)
PART A: MASK ALIGNMENT
1. Determine the sample volume and
type of vessel you will be using for your
experiment.
2. Prepare enough 1X External Well Factor
solution to fill 96 wells with your sample
volume. Dilute the 10X EWFS to 1X with
water.
3. Pipette the sample volume into the
correct vessel(s) and seal with the
appropriate cap or tape. Load
vessel(s) into the thermal cycler sample
compartment.
4. With the thermal cycler and detection
system on and the software open, click
the Calibration tab.
5. Click on the Mask tab (it will be
highlighted in green).
6. Click on Home, then highlight the Filter
Position 2 option.
7. Click on Take an Exposure, then click
on Show Mask.
8. If any pink pixels are present in the
mask, reduce the exposure time and
retake an exposure. Keep reducing the
exposure time and retaking the
exposure until no pink pixels are
present.
9. Click Optimize Mask, then click Save
Mask.
For example, this could be 50 µL sample
volume in a 96-well plate with sealing tape,
or it could be 25 µL sample volume in strip
tubes with flat caps.
Prepare a volume in excess of what is
actually needed so you do not run out.
A multichannel pipettor is a useful tool for
filling the plate or tubes.
Wear gloves when handling optically clear
plastics to avoid leaving fingerprints.
93
10. Remove well factor plate, tubes or
strips and save for Part C.
PART B: BACKGROUND CALIBRATION
1. Load an empty sealed plate, or
capped strips or tubes into the sample
compartment to fill it completely.
2. Select the Background tab.
3. Select a seal type and a vessel type,
and click on Collect Background
Readings. The software will launch a
short run that will last about 10 minutes.
4. When the background run is complete,
remove the empty vessel(s) and save in
a plastic bag until this calibration needs
to be repeated.
PART C: WELL FACTOR CALIBRATION
1. Place the external well factor plate or
tubes in the sample compartment.
2. Select the Well Factor tab.
3. Select a sample volume, a seal type,
and a vessel type.
4. Click Collect Persistent Well Factors. The
software will launch a short run that will
last about 10 minutes.
5. When the PWF run is complete, remove
the vessel(s) and save or discard. The
instrument is now ready for quantitative
PCR.
The 1X well factor solution has a shelf life of
one month. 10X solution keeps in the -20
freezer for a year.
94
SOP PCR-004
APPLIED BIOSYSTEMS 2720 THERMAL CYCLER
Revision A, February 22, 2011
Title: Operation of Applied Biosystems 2720 Thermal Cycler
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for operation of the AB 2720 thermal cycler.
2.0 SUMMARY OF METHOD
MATERIALS


AB 2720 thermal cycler
PCR reaction tubes, strips, or 96-well
plate
PART A: OPERATION
1. Plug in the thermal cycler and turn it
on. You will see a Home screen with the
time, date, and temperature at the top
and RUN, CREATE, EDIT, UTIL, and USER
at the bottom.
2. Use the F1-F5 softkeys to select one of
the options at the bottom of the
screen:
a. F1 to RUN an existing protocol
b. F2 to CREATE a new protocol
c. F3 to EDIT and existing protocol
3. Run a protocol that is already saved.
Choose the protocol you want with the
arrow keys and hit the softkey under
VIEW to check the program. If the
program is correct, hit the softkey
under START to begin. You must enter
the volume of your reaction mixture
before the program will begin. At this
step, open the lid and insert your
reaction tubes, strips, or plate into the
sample block, close the lid, and
proceed.
4. Create a new protocol. Use the arrow
keys to move through the screen and
use the number keys to enter the
appropriate times and temperatures.
When you are finished you may SAVE or
IMPORTANT: this instrument will only hold 0.2
mL sample tubes. If your reaction is in a 0.5
mL tube, you MUST transfer it to a 0.2 mL
tube.
95
START.
5. Edit an existing protocol. Choose the
protocol you want with the arrow keys
and hit the softkey under VIEW to edit.
Make the necessary changes and
SAVE.
6. When you have started a protocol the
instrument will warm up to the start
temp and then begin the program.
There is no pause before the program
starts, as there is with some other
instruments, so make sure your samples
are in the block before starting.
7. Every protocol has an automatic hold
at 4 degrees at the end of the
protocol, so your samples will be chilled
after the run. Still, you should attempt to
remove them as soon as possible after
the run and freeze them.
96
SOP PCR-005
CFX96 Real-time qPCR System
Revision B, April 11, 2011
Title: Use and Maintenance of CFX96 Real-time qPCR System
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for use and maintenance of the Bio-Rad CFX96 realtime qPCR system.
2.0 INTRODUCTION
The CFX96 is the newest qPCR instrument from Bio-Rad. It uses LED technology to excite up to 5
fluorophores in each well to monitor and quantify PCR products. The major benefit of this
instrument is that it allows the user to quantify the PCR products below cycle 30 (above which
the results are considered unreliable).
A great feature of the CFX96 is that the user does not need to do any time-consuming
calibration steps or plate setups before their PCR run. There are internal diagnostic procedures
that make calibration unnecessary.
Reading an entire 96-well plate takes only 3 seconds if using one channel (12 seconds if using all
five channels). The instrument automatically reads all wells.
The instrument is controlled by CFX Manager software, but it can be used without the software
as well. The software can be operated (protocol and plate design and data analysis) without
the instrument connected. The software works on Windows XP and above.
Data can be exported to an HTML or Excel file on the computer or directly onto a USB drive if
using the instrument without a computer.
3.0 SUMMARY OF METHOD
MATERIALS
1. CFX96 real-time qPCR system
2. Computer with CFX Manager
software
3. Optically clear plates for PCR;
sealing film
4. qPCR master mix reagent
PART A: OPERATION
To perform a qPCR run, follow these steps:
 Design a PCR experiment with
thermal program, plate setup,
data collection, and a melt curve
 Pipette reaction mixtures into a
plate
 Load plate into the sample block
and start the experiment
1. Open the CFX Manager software on
97
the attached computer. The Startup
Wizard will appear. You can choose to
create a new experiment, repeat an
existing one, or open data files.
Choose Create a new experiment.
Alternatively, instead of using the
Wizard you can select this option from
the File menu.
2. In the Protocol tab, you can select an
existing protocol and use or edit it, or
create a new one. If you have chosen
to edit or create a new protocol, you
will open the Protocol Editor. With this
tool, you can edit the temperature,
dwell time, and number of repeats of
each step of a protocol. Click on the
value you want to change in either
the graphic or in the text view and
enter the desired value. You can also
insert or delete steps using the buttons
to the left.
3. The protocol template that opens
when you create a new protocol
already includes a plate read step. To
add a plate read, click the step you
want, and then click the Add Plate
Read to Step button.
4. For a melt curve, click the Insert Melt
Curve button. Edit the melt
temperature range or increment time.
5. In the Plate tab, you can select an
existing plate setup and use or edit it,
or create a new one. The plate file
contains a description of the contents
of each well, the scan mode, and the
plate type. To run a real-time
experiment, the plate setup must
include at least one well with a
sample type and fluorophore, and you
must select a scan mode and a plate
size and type. Everything else about
the plate setup can be altered before,
during, or after the run.
6. The Plate Editor Window displays the
plate layout and provides tools to edit
it. Go to Settings in the Menu Bar to set
the plate size (number of wells) and
The Experiment Setup Window includes
three tabs: Protocol, Plate, and Start Run.
The quantitation (read) step should always
be after the extension step of PCR.
The melt curve portion of the program has
automatic settings that increase the temp in
0.5 degree increments from the annealing
temperature up to 95 degrees.
The instrument always reads all wells, so
there is no need to design a plate layout
before the run.
You can also scan in All Channels if you are
using more than one fluorophore, or a
different one than SYBR/FAM.
98
plate type (color). Choose 96-well and
clear.
7. In the Scan Mode drop menu, choose
SYBR/FAM only.
8. To label wells, first select them by left
clicking on the graphic. You can click,
hold and drag to select multiple wells.
9. Load the Sample Type first. Select a
Sample Type from the drop menu,
choosing from the following:
 Unknown: an experimental sample
you don’t know the concentration
of
 Standard: a sample of known
concentration
 NTC: no (DNA) template control
 Positive control: control reaction
with target DNA added
 Negative control: control reaction
without the target DNA added
(may have other DNA template
added)
10. Click a Load box to add a fluorophore
to the selected wells. You can also
specify a target for one or more
fluorophores, but this is only necessary
when multiplexing or if the data will be
used later for a gene study.
11. Sample Name can be skipped unless
you are compiling the data for a gene
study.
12. To enter a series of standards to
generate a standard curve for
quantitation, you need to select the
wells that contain the series and add
the fluorophore and sample type
(standard). Then load them as
replicates by checking the load box
next to Replicates. Click the Replicate
Series button and an editor will open.
Leave the Replicate Number and
Starting Replicate # at 1. This will
number the series starting with Std-1.
Choose horizontal or vertical
(depending on if you did the
standards in a row or a column). Click
99
Apply.
13. Click the Load check box next to
Concentration and enter the
concentration of the first standard.
Click the Dilutions Series button. Enter
all the information, including the
starting and ending replicate
numbers, the dilution factor, and
whether the concentrations are going
up or down as the replicate numbers
go up. Click Apply.
14. Repeat for any other standard series
you have on the plate, but change
the starting replicate number to
distinguish the series from each other.
Do NOT force the lid back—it operates via a
motor and you need only to touch the
button to make it move back and forth.
Do NOT block the lid from opening or
closing. Make sure there is clearance
behind and above the instrument for the lid
to open, and when you are closing the lid,
make sure there is nothing between the lid
and the base that would prevent it from
closing.
Our sample block is designed for plates with
0.2 mL wells only.
Optically clear plates and sealing film MUST
be used in this instrument. Do NOT use strips
and caps because the vessels need to be
low-profile or the optics will not function
correctly.
15. After editing or creating a new plate,
save it and move to the Start Run tab.
In the Start Run tab, you can set the
run settings and begin the run.
16. The Start Run tab lists the protocol and
plate setup you are going to use and
has a Start Run button that you click
when you are ready to run. When you
are ready to load your plate into the
sample block, press the Open Lid
button on the software’s Start Run tab,
or press the lid button on the front of
the instrument to open. Be patient, as
the lid opens slowly at first.
17. Place the sealed plate on the sample
block with well A1 in the upper left of
the block. Click the Open Lid button in
the software or press the lid button on
the front of the instrument to close the
lid.
18. With the lid closed, click the Start Run
button in the Start Run tab. You will be
prompted to save the name of the
data file and then the Run Details
Window will open. Here, you can
monitor the progress of the run. The
Run Details Window has three tabs:
Run Status, Real-time Status, and Time
Status.
 The Run Status tab shows the
status of the protocol and
DO NOT leave the unit holding at 4 degrees
overnight, since condensation can form on
the optics and damage them. Remove
amplicons promptly from the unit after the
run is over.
100


allows you to open the lid,
pause or stop the run, and skip
or repeat steps.
The Real-time Status Tab shows
the real-time fluorescence
data as they are collected.
The Time Status tab shows a
full-screen countdown timer for
the protocol.
19. When the run has finished, open the
lid, remove the plate, and close the
lid. Turn off the instrument and close
the software.
PART B: DATA ANALYSIS
1. Open a data file by going to File and
Open Data File. The Data Analysis
window will open. Click the
Quantitation tab to see an
amplification chart and standard
curve(s). Click on wells in the plate
graphic to select or deselect wells to
display data for.
2. The software automatically subtracts
the baseline from well data. You can
change this manually by going to
Settings and Baseline Threshold.
3. The cycle threshold line is calculated
automatically by the software, but you
can change this by dragging it up or
down using the mouse.
4. To create a report, click the Report
icon on the toolbar of the Data
Analysis window. Click checkboxes in
the report options list to include
specific information. Check the
preview on the right side of the screen
before saving or printing.
5. To export data files into Excel, right
click on the table and selecte Export.
PART C: TROUBLESHOOTING
Experiment Design
If the Experiment Setup Window does not
show a Plate tab, then the Protocol
You can view your melting data in two
formats: melting curve (RFU vs temperature)
and melt peak (the first derivative of the
melt curve).
The overall negative slope of melt curves is
due to the buffering capacity of the mixture
changing with temperature.
You’re interested in whatever melts above
80 degrees C. A rule of thumb is that the
melting temperature increases 2 degrees C
per base pair.
101
selected does not have a plate read step.
Add a plate read step to the protocol
and the Plate tab will appear.
PART D: MAINTENANCE
The LEDs have a 20-year lifetime so should
almost never have to be replaced.
If you change the plate type (from clear
to white) you will be asked to calibrate
the system using that plate and the
selected fluorophore.
102
SOP PHM-002
OPERATION AND MAINTENANCE OF ACCUMET MODEL AB15 pH METER
Revision A, June 8, 2011
Title: Operation and standardization of AB15 pH meter
1.0 SCOPE AND APPLICATION:
This SOP outlines the procedure for the proper operation and standardization of the AB15 pH
meter.
2.0 SUMMARY OF METHOD
MATERIALS







COMMENTS
AB15 pH meter
pH 4, 7, and 10 standard buffer
solutions (if calibrating)
50-mL beakers
pH probe with electrode
Deionized water in wash bottle
Waste beaker (250-500 ml)
Labeling tape and sharpie
PART A: STANDARDIZATION
1. Turn on pH meter and allow it to warm
up for at least 5 minutes. The probe
must be supported by the holder.
2. Select two standards that bracket the
expected pH of the solution to be
measured. If basic, use standards of
pH 7 and 10. If acidic, use standards of
pH 4 and 7. If the expected pH is
unknown, use standards of pH 4, 7, and
10.
The tip of the probe should be immersed in
a storage solution but not touching the
bottom of the vessel (this can damage the
probe).
Do not keep a pH probe out of a solution
for longer than a minute; it may damage
the probe. Do not turn probe upside
down.
3. Label 50-mL beakers with the pH of the
selected calibration standards. Pour 2535 ml of standard buffers into the
appropriately labeled beakers.
4. Check that the pH meter is in pH mode
(on the top left of screen); if not, press
and release the mode key until the
meter is in pH mode.
5. Completely rinse the probe with
deionized water (over the waste
collection beaker) and immerse the
electrode 1 cm in a buffer. Wait for the
The tip of the probe should not be
touching any surface.
103
reading to stabilize, and if it is greater
than ±0.03 pH units from the true pH
value, then you must standardize the
meter. If not, proceed to PART B.
6. Press the setup key twice and then the
enter key to clear the existing
calibration.
7. Press the std key to access
Standardization mode. The selected
buffer group will appear (2, 4, 7, 10, 12).
8. Press std again to initiate
standardization. The meter will
recognize the buffer and flash the pH
value on the screen. When the stable
icon appears, proceed to step 9.
9. Completely rinse the probe with
deionized water (over the waste
collection beaker) and immerse the
electrode 1 cm in the other buffer.
10. Repeat steps 7, 8, and 9 with this buffer
and a third if necessary.
11. Completely rinse the probe with
deionized water (over the waste
collection beaker), and immerse the tip
of the electrode into a storage solution
in a 50 mL Erlenmeyer flask.
PART B: OPERATION
1. Check that the meter is in pH mode (on
the top left of the display) and if it is
not, press and release the MODE key
until pH appears on the display.
2. Completely rinse the probe with
deionized water (over the waste
collection beaker) and immerse the
electrode 1 cm in the sample. Wait for
the reading to stabilize.
3. Record the pH measurement and
remove the probe from the sample.
Rinse thoroughly with deionized water
(over the waste collection beaker) and
replace in the storage solution.
When the meter accepts the second
buffer, it will display the percent slope
associated with the electrode’s
performance prior to returning to the
measure mode. If the electrode is with in
the range of 90-102%, the GOOD
ELECTRODE message will appear. If the
electrode is outside this range, the meter
will display ELECTRODE ERROR message and
will not return to the measure screen until
the user presses the enter key.
Add any comments, such as an error
message on the meter, low fill solution in
the probe, or low or contaminated probe
storage solution, to the appropriate log
sheet found in the equipment log book.
The meter will permit the user to use an
electrode outside the recommended
range, but the ERROR message will still
show.
Try recalibration (steps 6-10), if you get an
ELECTRODE ERROR message. If you get this
error message again after the second
calibration, report the electrode failure
immediately to your supervisor and enter
this on the instrument log sheet.
104
105
APPENDIX TO SOP PHM-002
pH PROBES FOR THE ACCUMET MODEL AB15 pH METER
The selection of the appropriate pH electrode (probe) to use with a pH meter can have an
impact on the quality of measurements and on the life of the equipment.
As of May 14, 2010, we are using Fisher Scientific accumet Liquid-Filled Mercury-Free pH/ATC
Epoxy Body Combination Electrodes (catalog# 13-620-631) with our pH meters. These have a
double junction with an Ag/AgCl electrode. They can be filled with saturated KCl solution
(catalog# SP138-500).
These electrodes are mercury-free, refillable, and are safe to use with Tris.
You may also see VWR SympHony refillable calomel electrodes (catalog# 14002-772). They can
be filled with calomel fill solution (catalog# 34108-024).
106
SOP RCV-001
RECEIVING MATERIALS
Revision A, April 21, 2011
Title: Receiving and Storing Materials
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for receiving packages and processing the materials
received.
2.0 SUMMARY OF METHOD
COMMENTS
PART A: RECEIVING
1. Locate the packing slip(s).
2. Find a department name, contact
name, or account code on the slip.
Ensure that this data corresponds to the
Biotechnology department. Our
account code is “55506”.
The full account number is a 16-digit
number and “55506” (or another account
code) will appear after “10-1-.”
3. If the shipment has been misdirected,
determine the correct recipient of it
and redirect.
Look for a contact name or department
on the packing slip, or call the warehouse
at 223-1042 to report.
4. Confirm that the packing slip and
actual inventory of shipment are in
agreement. Were all items received
and in the correct quantity?
If any items were reported shipped on the
packing slip, but were not in the shipment,
call the warehouse at 223-1042 to
investigate.
5. Open parcels and check for damaged
or missing items.
If items are damaged, non-functioning, or
incorrect, call the vendor immediately to
arrange for exchange or replacement. Be
sure to save all packing materials in order
to send the items back.
6. Locate the manual or product insert for
each product and follow any
unpacking, assembly, or storage
directions.
7. Manuals and product inserts can be
stored with the product (if feasible), or
in the file cabinet under the name of
the vendor or manufacturer.
8. If the item is a capital asset (generally,
over $500), write down the following
information: manufacturer, model,
serial number, location (campus,
building and room number). Send this
to the ACC Asset Management
107
department.
9. Perishable items should be stored at the
recommended temperatures. Before
storing, note the month and year of
receipt on the label of the item.
10. Non-perishable items can be stored in
the documented storage location (see
Equipment Locator).
11. Record the new item on the refrigerator
or freezer log, if applicable. Be sure to
note the date it arrived.
108
SOP SOL-001
LABELING SOLUTIONS
Revision A, January 17, 2008
Title: Labeling Prepared Solutions
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for labeling solutions prepared by students, faculty
and staff.
2.0 SUMMARY OF METHOD
COMMENTS
1. Obtain a solution prep form from the
instructor or from the lab form files.
2. Complete any calculations and enter
them at the bottom of the solution prep
form (or reference a page in your lab
protocol).
3. Obtain all chemicals, glassware, and
equipment needed to prepare the
solution and assemble so they are all
within reach.
4. Make a label for the vessel in which
you will prepare the solution; use
whatever name you would usually call
the solution (for example, “1X TAE
buffer”). Leave plenty of room for
information that will be added later.
Affix the label to the vessel before
adding any chemical.
Any solution or chemical must be labeled
with its identity if it is removed from its
primary container (i.e. the stock bottle).
5. Think of a unique ID number (control
number) for the solution. The format of
control numbers in our labs should
follow the following example:
The purpose of control numbers is for any
person working in the lab to discriminate
between any two solutions, regardless of
how similar they are, by the control
number.
TAE-EG-041106
The first three letters are an
abbreviation of the common name of
the solution (in this example, TrisAcetate-EDTA buffer). The second two
letters are the initials of the person (or
one of the people) who prepared the
solution (Evelyn Goss). The last six digits
are the date (always YYMMDD; in this
example, November 6, 2004).
In the unusual case that one individual
makes the same solution more than once
in one day, simply insert numbers after the
first three letters to indicate the order in
which they were made.
For example, the second batch of TAE
buffer that Evelyn Goss made on
November 6, 2004 would have the control
number:
TAE-2-EG-041106.
109
6. Fill out all sections of the solution prep
form (see Appendix A for an example).
If a field contains information that is not
applicable, enter “N/A” in that field.
Enter all information (except the
amount of each chemical used)
before preparing the solution. Enter the
amounts used after weighing and
delivering the chemical(s) to the
preparation vessel.
7. After preparing the solution and
transferring solution and label to the
storage vessel (if necessary), complete
the label by including the following
information:
Solution prep control number
Any special instructions, such as
expiration date or storage conditions
Safety warnings if the solution contains
hazardous materials
Course number and semester (if
applicable)
8. File the solution prep form
alphabetically in the top drawer of the
black file cabinet. If you are a student
taking a course, hand into your
instructor, who will make copies as
needed and give the original back to
you for your lab report. The instructor
should always keep a copy on file in
the lab.
Never enter the amount of a chemical
used until you have actually measured and
added the quantity. Do not enter the
calculated mass or volume; only enter the
actual, measured quantity.
110
Appendix A
SOLUTION PREP FORM
Name of Solution/Media:
Amount prepared:
Control # TAE-EG-041106
50X TAE Buffer
1L
Preparer(s):
Evelyn Goss
Component
Brand/lot #
(Vendor)
Trizma base
Preparation Date: November 6, 2004
FW or initial
concentration
Amount
used
Final
concentration
Sigma/022385
Storage
conditions/
date received
RT/031505
121.1 g/mol
242 g
2M
Glacial acetic acid
EMD/43DBB31
RT/053099
60.05 g/mol
57.1 mL
5.71%
EDTA
Fisher/
960654A
RT/012502
372.24 g/mol
37.2 g
100 mM
Balance used
#5
Calibration status
OK
pH meter used
N/A
Calibration status
N/A
Initial pH
8.5
Final pH
8.5
Adjusted pH with
N/A
Prep temperature
RT
Sterilization procedure/
sterility testing
N/A
Storage conditions
RT
Calculations/Comments:
See protocol on page A1-31 of Short Protocols in Molecular Biology, Vol.2, 5 th Ed., Ausubel, et al.
111
SOP SPC-001
GENESYS 10 SPECTROPHOTOMETER
Revision A, June 15, 2011
Title: Use and Maintenance of the Genesys 10 Spectrophotometer
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for use and maintenance of the Genesys 10
Spectrophotometer.
2.0 SUMMARY OF METHOD
PART A: BASIC ABSORBANCE, TRANSMITTANCE,
AND CONCENTRATION READINGS
1. Turn on the spectrophotometer using the
power switch on the back of the
instrument.
2. Wait for the instrument to initialize and
complete internal tests.
3. From the home screen, press the softkey
at the bottom right labeled “Basic ATC.”
4. Press the softkey labeled “Set nm.” Enter
the desired wavelength using the numeric
keypad and press Enter.
5. Choose cuvettes appropriate for the
wavelength. Make sure they are clean on
the inside and outside and free from
scratches or stains.
6. Wearing gloves, fill one clean cuvette at
least halfway with the solution to be used
as a blank.
7. Fill another cuvette at least halfway with
the solution to be tested for absorbance.
If there is a shortage of cuvettes, the blank
can be measured first, then removed, and
the blank solution replaced with the
solution to be tested.
8. Carefully wipe the optically clear sides of
the cuvettes with a Kimwipe or lens paper
to remove any drops of liquid, dust, or
fingerprints.
This option will allow the user to make basic
absorbance measurements at a given
wavelength.
Some plastics are not UV-transparent and
would not be useful for UV absorbance
readings.
The blank solution should be identical to or as
similar as possible to the solution in which the
compound to be measured is suspended. This is
usually water or an aqueous buffer.
Orient the cuvette such that the optically clear
sides are aligned with the light path.
It is best to handle cuvettes by the optically
opaque sides to avoid leaving any residue on
the clear sides.
112
9. Open the sample compartment. Insert the
blank cuvette in the sample holder
labeled “B”. Orient the cuvette such that
the optically clear sides are aligned with
the light path.
10. Insert the sample cuvette in the first
sample holder, labeled “1”. If there are
additional samples and enough cuvettes
to hold them, insert these in the other four
sample holders and note their positions.
11. Close the sample compartment.
12. Locate the numbered sample selection
keys (arranged in a circle on the keypad).
Press “B” to select the blank.
13. Press the softkey labeled “Measure blank”.
The display should read 0.000A.
14. Press the sample selection key with the
number of the sample you wish to
analyze. The sample holder carousel will
rotate and the instrument will analyze the
selected sample, displaying the
absorbance.
15. If the absorbance is greater than 2.0, the
display will show an error message stating
that the sample is outside the range of the
instrument. This will require you to dilute
the sample to gain an accurate reading.
16. Repeat steps 14-15 with all other samples.
17. When you are finished, clean the cuvettes
by first emptying them, then flushing with
several changes of purified water. Next,
flush with several changes of 70-95%
ethanol. Finally, dry upside down on a
paper towel before storing in a safe
place.
PART B: DNA ANALYSIS BY SCANNING
1. From the main menu, highlight “Nucleic
Acid Tests” and press Enter.
2. Select “DNA with Scan 260/230”.
3. Parameters will display; press the softkey
labeled “Run Test”.
A 1/10 dilution is a good place to start. Use the
blank solution to dilute the sample. If the
absorbance is still too high, dilute again and
repeat until an accurate measurement is
obtained. Then, multiply the absorbance
reading by the dilution factor to obtain the
absorbance of the original concentration.
Never use any abrasive cleansers, soaps, or
brushes on cuvettes. This can scratch them or
leave a contaminating residue.
Disposable plastic cuvettes can be used a few
times before they become scratched or stained
and need to be discarded. Clean these
carefully and they will last for several uses.
Quartz cuvettes look like glass and are made to
have a long life. Treat these with special care
because they are very expensive. They will last
many years if used carefully.
113
4. Insert a cuvette filled with the blank
solution appropriate to your experiment
into the sample holder labeled “B”.
5. Press the softkey labeled “Collect
Baseline” and the instrument will collect
baseline data.
6. When baseline collection is complete,
remove the blank cuvette.
7. Insert a cuvette filled with your sample into
one of the numbered sample holders.
8. Press the softkey labeled “Collect Data”
and the instrument will scan the sample.
9. When complete, the instrument will print a
record of the scan. Collect the printout
and press Escape to return to the main
menu.
10. Remove all samples from the sample
compartment and clean the cuvettes
thoroughly (see 17, Part A).
11. If you are the last to use the
spectrophotometer, power off the
instrument.
PART C: KINETICS
1. From the main menu, select “Kinetics” and
press Enter.
2. Select “Stored Tests” and press Enter to
recall a stored test. Or, change the
parameters on the Kinetics home screen
to create a new test, then press the “Save
Test” softkey to save it.
3. With the selected test open, press the
“Run Test” softkey to start the test.
4. Insert a cuvette filled with the blank
solution appropriate to your experiment
into the sample holder labeled “B”.
5. Press the softkey labeled “Collect
Baseline” and the instrument will collect
baseline data.
114
6. When baseline collection is complete,
remove the blank cuvette.
7. Insert a cuvette filled with your sample into
one of the numbered sample holders.
8. Press the softkey labeled “Collect Data”
and the instrument will begin the test.
9. If you did not select Auto Print while
defining parameters for the test, you will
need to press the Print button to obtain a
printout of your results.
115
SOP SPC-002
BioMate 3 SPECTROPHOTOMETER
Revision A, June 15, 2011
Title: Use and Maintenance of the BioMate 3 Spectrophotometer
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for use and maintenance of the BioMate 3
Spectrophotometer.
2.0 SUMMARY OF METHOD
MATERIALS


BioMate 3 Spectrophotometer
Cuvettes
PART A: BASIC ABSORBANCE, TRANSMITTANCE,
AND CONCENTRATION READINGS
1. Turn on the spectrophotometer using the
power switch on the back of the instrument.
2. Wait for the instrument to initialize and
complete internal tests.
3. From the home screen, press the softkey at
the bottom right labeled “Basic ATC.”
4. Press the softkey labeled “Set nm.” Enter the
desired wavelength using the numeric
keypad and press Enter.
5. Choose cuvettes appropriate for the
wavelength. Make sure they are clean on
the inside and outside and free from
scratches or stains.
6. Wearing gloves, fill one clean cuvette at
least halfway with the solution to be used as
a blank.
7. Fill another cuvette at least halfway with the
solution to be tested for absorbance. If
there is a shortage of cuvettes, the blank
can be measured first, then removed, and
the blank solution replaced with the solution
to be tested.
8. Carefully wipe the optically clear sides of
the cuvettes with a Kimwipe or lens paper
This option will allow the user to make basic
absorbance measurements at a given
wavelength.
Some plastics are not UV-transparent and
would not be useful for UV absorbance
readings.
The blank solution should be identical to or as
similar as possible to the solution in which the
compound to be measured is suspended. This
is usually water or an aqueous buffer.
It is best to handle cuvettes by the optically
116
to remove any drops of liquid, dust, or
fingerprints.
opaque sides to avoid leaving any residue on
the clear sides.
9. Open the sample compartment. Insert the
blank cuvette in the sample holder. Orient
the cuvette such that the optically clear
sides are aligned with the light path.
10. Close the sample compartment.
11. Press the softkey labeled “Measure blank”.
The display should read 0.000A.
12. Remove the blank cuvette and insert a
cuvette filled with your sample. The
instrument will display the measured
absorbance.
13. If the absorbance is greater than 2.0, the
display will show an error message stating
that the sample is outside the range of the
instrument. This will require you to dilute the
sample to gain an accurate reading.
14. Repeat steps 14-15 with all other samples.
15. When you are finished, clean the cuvettes
by first emptying them, then flushing with
several changes of purified water. Next,
flush with several changes of 70-95%
ethanol. Finally, dry upside down on a
paper towel before storing in a safe place.
16. If you are the last to use the
spectrophotometer, power off the
instrument.
PART B: DNA ANALYSIS BY SCANNING
1. From the main menu, highlight “Nucleic
Acid Tests” and press Enter.
2. Select “DNA with Scan 260/230”.
3. Parameters will display; press the softkey
labeled “Run Test”.
4. Insert a cuvette filled with the blank solution
appropriate to your experiment into the
sample holder.
A 1/10 dilution is a good place to start. Use
the blank solution to dilute the sample. If the
absorbance is still too high, dilute again and
repeat until an accurate measurement is
obtained. Then, multiply the absorbance
reading by the dilution factor to obtain the
absorbance of the original concentration.
Never use any abrasive cleansers, soaps, or
brushes on cuvettes. This can scratch them or
leave a contaminating residue.
Disposable plastic cuvettes can be used a
few times before they become scratched or
stained and need to be discarded. Clean
these carefully and they will last for several
uses.
Quartz cuvettes look like glass and are made
to have a long life. Treat these with special
care because they are very expensive. They
will last many years if used carefully.
117
5. Press the softkey labeled “Collect Baseline”
and allow the instrument to collect baseline
data.
6. When baseline collection is complete,
remove the blank cuvette.
7. Insert a cuvette filled with your sample into
the sample holder.
8. Press the softkey labeled “Collect Data”
and allow the instrument to scan the
sample.
9. When complete, the instrument will print a
record of the scan. Collect the printout and
press Escape to return to the main menu.
10. Remove the sample from the sample
compartment and clean all cuvettes
thoroughly (see 17, Part A).
11. If you are the last to use the
spectrophotometer, power off the
instrument.
PART C: KINETICS
1. From the main menu, select “Kinetics” and
press Enter.
2. Select “Stored Tests” and press Enter to
recall a stored test. Or, change the
parameters on the Kinetics home screen to
create a new test, then press the “Save
Test” softkey to save it.
3. With the selected test open, press the “Run
Test” softkey to start the test.
4. Insert a cuvette filled with the blank solution
appropriate to your experiment into the
sample holder labeled “B”.
5. Press the softkey labeled “Collect Baseline”
and the instrument will collect baseline
data.
6. When baseline collection is complete,
remove the blank cuvette.
7. Insert a cuvette filled with your sample into
118
one of the numbered sample holders.
8. Press the softkey labeled “Collect Data”
and the instrument will begin the test.
9. If you did not select Auto Print while defining
parameters for the test, you will need to
press the Print button to obtain a printout of
your results.
119
SOP SPC-003
NANODROP 1000 SPECTROPHOTOMETER
Title: Operation of NanoDrop 1000 Spectrophotometer
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for operation of the NanoDrop 1000
Spectrophotometer.
2.0 SUMMARY OF METHOD
MATERIALS



NanoDrop 1000 spectrophotometer
Attached computer with ND1000
software
Kimwipes, DI water, p2 pipette
PART A: OPERATION
IMPORTANT: do not handle the fiber optic
cable on the NanoDrop. This can damage
it and cause problems with the instrument.
When lifting and lowering the pedestal,
handle only by the metal part at the
bottom. Keep the instrument away from
objects that could fall on or snag the cable
to prevent damage.
1. Open the ND1000 software on the
attached computer and select the
analysis module of interest (nucleic
acid, protein, etc).
2. Follow prompts to initialize the
spectrophotometer by pipetting 2 uL of
DI water onto the bottom pedestal and
clicking OK. When this is finished, wipe
the water off with a kimwipe.
3. Establish a reference or blank using the
appropriate buffer. Pipette 1-2 µL of
blanking buffer onto the bottom
pedestal and click the Blank button.
Wipe the blank off with a kimwipe.
4. Pipette 1-2 µL of the sample onto the
bottom pedestal and click the Measure
button. Wipe the sample off with a
kimwipe.
5. Measure other samples by repeating
steps 3 and 4.
6. If desired, pipette 1-2 µL of deionized
water onto the bottom pedestal and
wipe with a Kimwipe to clean the
sample holder.
DO NOT use detergents or alcohol to clean
the pedestals, as their use may result in
pedestals becoming unconditioned.
120
SOP SPC-004
LAMBDA BIO+ SPECTROPHOTOMETER
Revision A, June 8, 2011
Title: Use and Maintenance of the Lambda Bio+ Spectrophotometer
1.0 SCOPE AND APPLICATION:
This SOP outlines the procedure for use and maintenance of the Lambda Bio+
Spectrophotometer.
2.0 SUMMARY OF METHOD
MATERIALS


Lambda Bio+ Spectrophotometer
Cuvettes with 15 mm sample window
NOTE: The beam height of this instrument is 15 mm.
You must either fill the cuvette at least 15 mm high,
or use a 15 mm height adaptor. When using a UV
wavelength, you must use UV-transparent cuvettes.
PART A: SINGLE WAVELENGTH READINGS
1. Turn on the spectrophotometer by pressing the
power button.
2. Wait for the instrument to initialize and complete
internal tests. The default home page will be
displayed.
Some plastics are not UV-transparent and
would not be useful for UV absorbance
readings. Quartz cuvettes are UVtransparent.
Make sure that the sides of the cuvette
facing the sides of the instrument are
optically transparent. The light path
travels from right to left across the
instrument.
It is best to handle cuvettes by the
optically opaque sides to avoid leaving
any residue on the clear sides.
3. Press 1 on the number keypad to choose Single
Wavelength.
4. Enter the wavelength you would like to use,
then press the green ► button on the keypad to
accept.
5. Insert a cuvette containing the appropriate
blank solution. Press the 0A/100%T button on the
keypad to take a blank reading.
6. Remove the blank cuvette and insert a cuvette
containing the sample. Press the green ►
button to take a reading. The absorbance will
be displayed.
7. If the absorbance value of a sample is above
the range of the instrument, you will need to
dilute the sample.
8. Repeat with any other samples.
The blank solution should be identical to
or as similar as possible to the solution in
which the compound to be measured is
suspended. This is usually water or an
aqueous buffer.
A 1/10 dilution is a good place to start.
Use the blank solution to dilute the
121
9. When you are finished, clean the cuvettes by
first emptying them, then flushing with several
changes of purified water. Next, flush with
several changes of 70-95% ethanol. Finally, dry
upside down on a paper towel before storing in
a safe place.
10. If you are the last to use the spectrophotometer,
power off the instrument.
PART B: MULTI-WAVELENGTH READINGS
1. Follow the procedure for single wavelength
readings with these two modifications:
2. In Step 3, choose 2 for multi-wavelength
readings.
3. In Step 4, enter two or more wavelengths.
sample. If the absorbance is still too high,
dilute again and repeat until an accurate
measurement is obtained. Then, multiply
the absorbance reading by the dilution
factor to obtain the absorbance of the
original concentration.
Never use any abrasive cleansers, soaps,
or brushes on cuvettes. This can scratch
them or leave a contaminating residue.
Disposable plastic cuvettes can be used
a few times before they become
scratched or stained and need to be
discarded. Clean these carefully and
they will last for several uses.
Quartz cuvettes look like glass and are
made to have a long life. Treat these with
special care because they are very
expensive. They will last many years if
used carefully.
PART C: ABSORBANCE SPECTRUM
1. Choose 3 for Spectrum in the default home
page.
2. Enter the start and end wavelengths and press
the green ► button.
3. Insert a cuvette containing the appropriate
blank solution. Press the 0A/100%T button on the
keypad to take a blank reading.
4. Remove the blank cuvette and insert a cuvette
containing the sample. Press the green ►
button to take a reading. The absorbance
spectrum will be displayed.
5. Repeat with any other samples.
6. When you are finished, clean the cuvettes by
first emptying them, then flushing with several
changes of purified water. Next, flush with
several changes of 70-95% ethanol. Finally, dry
upside down on a paper towel before storing in
a safe place.
The blank solution should be identical to
or as similar as possible to the solution in
which the compound to be measured is
suspended. This is usually water or an
aqueous buffer.
7. If you are the last to use the spectrophotometer,
power off the instrument.
PART C: KINETICS
A 1/10 dilution is a good place to start.
122
1. From the Standard Methods home screen, press
6 for Kinetics.
2. Enter the wavelength, delay time (if any),
duration time, and interval length using the
numeric keypad and arrows.
3. Press the green ► button to continue.
4. Select the measurement mode using the left
and right arrows (you will most likely want to
select “Slope”). Press the down arrow to get to
units, then enter the units manually or press the
Options button to choose from a list of units.
Press the down arrow and change the “Factor”
setting if you need to multiply results by a
dilution factor.
5. Press the green ► button to continue.
6. Insert a cuvette containing the reference
sample and press the 0A/100%T button to take
a blank reading.
7. Insert a cuvette containing the sample and
press the green ► button to begin measuring.
The window will display a real-time plot of
absorbance.
8. Press the Options button to print, save, or
change view settings.
Use the blank solution to dilute the
sample. If the absorbance is still too high,
dilute again and repeat until an accurate
measurement is obtained. Then, multiply
the absorbance reading by the dilution
factor to obtain the absorbance of the
original concentration.
Never use any abrasive cleansers, soaps,
or brushes on cuvettes. This can scratch
them or leave a contaminating residue.
Disposable plastic cuvettes can be used
a few times before they become
scratched or stained and need to be
discarded. Clean these carefully and
they will last for several uses.
Quartz cuvettes look like glass and are
made to have a long life. Treat these with
special care because they are very
expensive. They will last many years if
used carefully.
123
SOP SPC-008
NANODROP 2000 SPECTROPHOTOMETER
Revision A, June 8, 2011
Title: Operation of the NanoDrop 2000 Spectrophotometer
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for operation of the NanoDrop 2000
Spectrophotometer.
2.0 SUMMARY OF METHOD
MATERIALS
 Nanodrop 2000 Spectrophotometer
 Attached computer with ND2000
software
 P2 or P10 pipettor and tips
 Deionized water and Kimwipes
PART A: Using the NanoDrop 2000
1.
Open the ND2000 software on the
desktop of the attached computer. If
asked for a password, click Enter without
typing a password.
2. The main menu will open and you will be
able to choose from a number of
methods, including Nucleic Acid, Protein,
etc. Select the appropriate method and it
will open.
3. You will be asked if you would like to open
the previously used workbook; choose No.
4. Make sure arm is down and click ok to
start the Wavelength Verification.
5. Go to File and New Workbook. Name and
save your workbook; all samples will now
be saved to your workbook until you
select a new workbook. This provides a
simple way to tabulate and report
multiple measurements.
6. Establish a reference or blank using the
appropriate buffer or water (whatever
your sample is dissolved in). Pipette 1-2uL
of blank solution onto the bottom
pedestal and click “BLANK”, located at
Note: the blank reading will not display any
values or a spectrum. These will not appear until
you perform a measurement. If you blank again
after taking a measurement, the data and
spectrum from the previous measurement will
remain on the screen.
To verify the blank, click Measure to record
your blank solution as a sample. A spectrum
124
the top left corner of the screen.
and absorbance and concentration values will
appear on the display.
7. Wipe upper and lower pedestals with a
Kimwipe.
8. Pipette 1-2uL of the sample onto the
bottom pedestal and click “MEASURE” to
obtain an absorbance. You may name
the sample in the Sample ID field.
9. Wipe upper and lower pedestals with a
Kimwipe.
10. Measure other samples by repeating steps
8 and 9.
11. If desired, pipette 1-2uL of deionized
water onto the bottom pedestal and wipe
with a Kimwipe to clean the sample
holder.
DO NOT use detergents or alcohol to clean the
pedestals, as this may result in pedestals
becoming unconditioned.
125
SOP TAC-001
TACHOMETER
Revision A, June 8, 2011
Title: Use and Maintenance of Tachometer
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for use of a tachometer to measure the speed of a
centrifuge.
2.0 SUMMARY OF METHOD
MATERIALS



Tachometer
Centrifuge
Reflective tape, Sharpie
PART A: SETUP
1. Locate the part of the centrifuge that
will show through the transparent
viewing window on the centrifuge lid.
Usually, this is the very center of the
rotor.
The tachometer works by shining a light
onto a visible, rotating part of the
centrifuge rotor. When there is a reflective
object on this part, the light will be
reflected back in bursts at the same
frequency as the rotor is moving (in rpm).
2. Open the centrifuge and observe this
part. If it is reflective, you will need to
color it black with a marker.
3. Attach a strip or triangle of reflective
tape to one side of the visible part.
PART B: OPERATION
1. Close the centrifuge and start a run.
2. Set the tachometer to Photo settting by
moving the toggle switch all the way to
the right.
3. Hold the tachometer with the light
beam shining down into the viewing
window. There is a foot on the
tachometer that will allow you to rest it
on the surface of the centrifuge.
4. Hold the Measure button (on the side
of the tachometer). Numbers will
appear on the LCD screen.
NOTE: the tachometer should be stored
without batteries because they become
run down if stored in the instrument. Please
insert batteries before using.
126
5. The reading should be approximately
the same as the speed setting on the
centrifuge.
6. When finished using the tachometer,
remove the batteries because they will
be run down if stored in the instrument.
PART C: MAINTANANCE
1. Once per year, the tachometer should
be sent to the Control Company for
calibration. See the calibration
document in the box for the address.
If the reading is twice the speed settting on
the centrifuge, you have most likely
situated the reflective tape so that two
burst of light are reflected per rotation. Fix
the tape so that only one burst of light will
be reflected per rotation.
127
SOP VAC-001
INTEGRA VACUSAFE ASPIRATOR
Revision A, August 16, 2011
Title: Integra VacuSafe Aspirator
1.0 SCOPE AND APPLICATION:
This SOP outlines the setup, use, and cleaning of the Integra VacuSafe Aspiration System for cell
culture.
2.0 SUMMARY OF METHOD
Materials
 VacuSafe aspiration system
 Sterile pipettes
Operation
1. Add approximately 100 mL of 10%
bleach to the VacuSafe bottle and
replace the lid.
2. Turn on the VacuSafe pump.
3. Grasp the VacuSafe controller and
insert a sterile pipette.
4. Place the tip of the pipette in the liquid
to be aspirated and depress and hold
the button on the side of the controller
to open the vacuum.
5. Liquid should flow into the tubing and
into the bottle. To stop aspirating,
release the button.
6. When finished, remove the pipette from
the controller. Flush the controller and
tubing with air by depressing the button
and allowing air to flow through the
system. Turn off the vacuum pump.
7. To empty the bottle, remove the small,
clear silicone cap from the third hold
on the lid of the bottle. This will allow air
into the system and you will be able to
unscrew the lid.
COMMENTS
Bleach is used in the bottle to kill any cells
so the flow-through will be
decontaminated before disposal.
Make sure the VacuSafe is somewhere
where it will not be knocked over, but is
convenient for use.
If the pipette is difficult to insert, try wetting
the end with alcohol.
128
SOP WPS-001
OPERATION AND CARE OF RoDi WATER PURIFICATION SYSTEM
Revision A, June 8, 2011
1.0 SCOPE AND APPLICATION:
This SOP outlines the policy and procedure for operation and care of the RoDi water purification
system.
2.0 SUMMARY OF METHOD
MODES OF OPERATION
1. If RoDi unit is in Run mode, you will see
a number (such as “17.6”) on the digital
display. This is the resistivity of the water
in megaohms•cm and indicates the
purity of the water. While in Run mode,
water is being purified continuously.
Unit should be in Run mode to dispense
water.
2. If RoDi unit is in Standby mode, you will
see “Sby” on the LED readout. The unit
should be left in Standby mode when
not in use to conserve the UV light bulb,
RO membrane and cartridges.
COMMENTS
The more pure the water, the higher its
resistivity will be.
In Standby mode, the pump will operate
for 10 minutes out of every hour, and the
UV light will operate for 10 minutes out of
every four hours.
3. If RoDi unit is in Idle mode, you will see
“IdL” on the LED readout. Idle mode
halts purification until Start/Stop is
pressed and unit enters Run mode. Idle
mode should be avoided to ensure a
supply of high purity water at all times.
PROCEDURE: OPERATION
1. Locate the container into which you
intend to dispense purified water.
2. Identify the operational mode of the
unit by using the guidelines in the
above section. If it is not in Run mode,
press Start/Stop. A number indicating
the resistivity of the water should
appear.
3. If necessary, allow the resistivity to rise
until water has reached the desired
purity. A resistivity of at least 16
megaohms•cm is adequate for most
Glass is the best storage material for water
containers, especially for long-term
storage, because it does not leach
materials like plastic does.
Unit should be left on at all times. If unit is
turned off, locate the power switch on the
back of the unit and turn on. Notify the lab
assistant if unit is turned off.
129
applications.
4. Position the container under the drawoff valve without touching the valve to
avoid contamination of the unit.
5. Press down on the draw-off valve lever
to dispense water. Lever will click down
and stay in position. When finished,
move the lever up to the off position.
6. Press Standby to put the unit in Standby
mode. This will slow the purification
processes to conserve the cartridges,
membrane and UV bulb, while still
ensuring a supply of high purity water
for when Run mode is activated again.
TROUBLESHOOTING
If an error message appears on the display
or if you encounter any problems with the
unit, see the troubleshooting guide starting
on page 41 of the manual.
MAINTENANCE
1. Every six months, the reverse osmosis
prefilter should be replaced. The
Replace RO Prefilter LED (small red
light) will turn on every six months.
2. If residual deposits are evident in
feedwater reservoir, or if a new 0.2
micron final filter clogs rapidly, the
reservoir should be cleaned according
to directions on page 28 of the manual,
and cartridges should be replaced.
3. When recirculated water will not rinse
up to desired purity level, replace
cartridges. If problem persists, clean the
resistivity cell according to directions on
page 30 of the manual.
4. If low RO purity LED is illuminated even
after an hour of rinse-up, replace the
RO membrane.
130
Record of Revisions to Standard Operating Procedures
Table 1: Updates in the 9th Edition, Fall 2012
SOP Title
Changes
Justification
CAM-001 (VistaVision
Microscope Camera
ELC-001 (Bio-Rad Mini-Sub
GT cells)
FCT-001 (Accuri Flow
Cytometer)
FLU-001 (Quantech
Fluorometer)
FLU-003 (NanoDrop 3300
Fluorospectrophotometer)
PCR-002 (Bio-Rad iCycler
for endpoint PCR)
PCR-003 (Stratagene
Mx4000)
PCR-006 (Bio-Rad iCycler
for Real-time qPCR)
SPC-005 (GE Nanovue
Spectrophotometer)
SPC-007 (Bio-Rad
SmartSpec)
ELC-004 (Edvotek
Electrophoresis Chambers
AUT-001 (VWR/Thermo
Scientific Sterilemax
Autoclave)
SOP removed.
Equipment no longer in use
in our labs.
Equipment no longer in use
in our labs.
Equipment rarely used in
our labs.
Equipment no longer in use
in our labs.
Equipment no longer in use
in our labs.
Equipment no longer in use
in our labs.
Equipment no longer in use
in our labs.
Equipment no longer in use
in our labs.
Equipment no longer in use
in our labs.
Equipment no longer in use
in our labs.
Equipment is frequently
used in our labs.
The same model Sterilemax
autoclave was sold by
VWR and Thermo Scientific
and we own one of each.
Renamed to remove
ambiguity.
A support technician from
Thermo suggested we use
deionized water instead of
tap.
Incorrect label was left
from using MT balance SOP
as a template.
BAL-005 (Ohaus Scout Pro
Balance)
SOP removed.
SOP removed.
SOP removed.
SOP removed.
SOP removed.
SOP removed.
SOP removed.
SOP removed.
SOP removed.
SOP added.
Title of SOP and
name of autoclave
changed from
“VWR” to
“VWR/Thermo
Scientific Sterilemax).
Changed “tap
water” to “deionized
water.”
Corrected name of
balance in the title
from “Mettler-Toledo
PL1502-S” to “Ohaus
Scout Pro SP402.”
Name of
Editor
Evelyn Goss
Evelyn Goss
Evelyn Goss
Evelyn Goss
Evelyn Goss
Evelyn Goss
Evelyn Goss
Evelyn Goss
Evelyn Goss
Evelyn Goss
Evelyn Goss
Evelyn Goss
Evelyn Goss