Biochemistry Practical
Zhihong Li (李志红) PhD
Dept.of Biochemistry
REGULATIONS OF PRACTICAL
Successful experiments usually depend on accurate
managements. All students should comply with these
regulations.
• 1.The students should come into the lab on time. It
is prohibit being absent, late or leaving earlier. Anyone
who is late for 15 minutes or more should be thought
as absenteeism.
2. It is necessary to put on the work clothes before
entering the lab. Not permitted to come into if the
students are wearing vest, short pants or slipper.
3. Keep quiet and neat in the lab. Any actions, which
are not concerned with your experiment, should be
avoided.
4. The students should know the content of
practical, and write the objectives, principle
and procedure on the report book in advance.
(you can download the content from our website)
5. Carefully observe the experimental phenomena,
record the experimental result and discuss the
significance of the experiment.
6. Wash your hands in time after finished the
experiment or touched the toxic chemicals.
7. Discard solid wastes in the provided waste
containers, not in the sink! Rinse non-hazardous,
water-soluble wastes such as small amounts of acids
down the drain.
8. Carefully and accurately operate the apparatus.
It should be kept in good order after your finishing
the operation. It should be compensated according to
the cause that results in the damage of the
apparatus.
9. Each student should clean the bench, take
back the apparatus to Room 2509, treat with the
trash, and leave off after the permission of the
instructor. The students who are on duty should
clean the lab after the experiment according to the
duty arrangement sheet.
10. The monitor should collect the report books
and hand over them to Room S-2514 (or S-2509)
before next experiment.
Contents
1
Volume transfer, accuracy and precision
2
Spectrophotometry & plotting of calibration
curve
3
Serum total protein
4
Enzyme inhibition test
5
Alkaline phophatase
6
Serum glucose test
7
Final experimental operation exam
Final practical grade
• Final practical grade=
– Daily performance (30%): report book,
attendance, operation and so on.
– Final operation exam (35%)
– Final writing exam (35%)
• 1/3 absent, can not take part in the
final exam.
BIO - 01
Volume transfer,
accuracy and precision
PURPOSE
 To learn how to handle pipettes,
micropipetts and electronic analytical balance.
 To learn how to dispense solution properly.
 To learn how to dispose and analyze the
experimental data correctly.
Part Ⅰ: PIPETTE
•Two types of pipette
are commonly used in
lab:
•Volumetric (or
transfer) pipets
•Measuring pipets
• Volumetric pipette has one calibration mark and is
designed to deliver one fixed volume.
• Measuring Pipets include Serological and Mohr
pipettes deliver various volumes to varying degrees of
accuracy. The measuring pipette is calibrated along
its length.
TECHNIQUES FOR USING PIPETTES
1. Pipettes should be used only with Rubber Bulbs,
mouth pipetting should not be allowed.
2. Squeeze the air out of the bulb and press the
opening of the bulb against the opening of the pipet.
Notice: the tip of the pipet must be kept under the
surface of the liquid the entire time suction is
applied or air will be sucked into the pipet.
3. Fill the pipette above the calibration mark using
a pipette bulb.
4. Quickly remove the bulb and place your index
finger (not your thumb) over the end of the
pipet..
5. Adjust liquid level to the calibrating line during
delivery. The lowest part of the meniscus, when
it is sighted at eye level, should be level with
calibration line on the pipette.
Right eye level
6. Tip the pipette against the beaker to remove
any excess solution.
7. Reset the pipette tip against the wall of the
container into which the solution is to be
transferred and allow the solution to drain.
Leave the pipette in this position for at least 10
seconds after all the solution appears to have
drained out and touch the pipet tip to the side of
the flask to remove any droplets.
8. Remove the pipet. DO NOT BLOW OUT THE
SOLUTION REMAINING IN THE PIPET.
The pipettes are calibrated in a manner that
takes into account the solution which remains at the
tip due to surface tension.
These pipettes are generally
used in handling reagents and are
considered not accurate enough to
handle standard and sample.
Part Ⅱ:
Micropipette
Multi-channel pipette
Single-channel pipette
FUNCTION OF THE MICROPIPETTE
• Micropipettes are used to transfer small
liquid volumes.
• It is a precision instrument calibrated in
microliters (μL).
1ml = 1000 (μL)
• It was invented in 1957 by Heinrich Schnitger
in Germany.
• These pipettes operate by piston-driven
air displacement.
CAREFUL HANDLING OF THE
MICROPIPETTE
• The micropipette must be handled with great
care, to avoid damage.
• A damaged micropipette may produce
imprecise measurements and this could
affect the results of the experiment.
• Do not rough-handle or drop the
micropipette.
Structure of micropipette
•Push botton
• handle
• tip ejector
• digital
volume
indicator
0.5-20µl
20-200µl
100-1000µl
tip
• tip ejector arm
• tip holder
How to handle micropipette:
Select proper Pipette:
Micropipette have different volume range.
P5000 1,000 ~ 5,000μl
P1000 200 ~ 1,000 μl
P200
50 ~ 200 μl
P100
20 ~ 100 μl
P20
2 ~ 20 μl
P10
0.5 ~ 10 μl
Micropipettes
• Micropipettes have 3 positions:
– Rest position.
– First stop.
– Second stop.
Operating the Micropipette
– 3 Volumes:
StepPipettors
1: Set
and read the Volume
Volume Adjustment Knob:
• Hold the micropipette in one hand and with the other
hand, turn the volume adjustment knob (or shaft) until
the volume indicator displays the desired volume.
•
IF THE VOLUME ADJUSTMENT KNOB IS HARD
TO TURN, STOP AND CALL THE INSTRUCTOR.
•
DO NOT TRY TO FORCE THE KNOB TO TURN
Digital Volume Indicator:
BECAUSE YOU WILL DAMAGE THE MICROPIPETTE.
•
Do not adjust the micropipette volume above or
below that recommended range (!!!).
Operating the Micropipette-2
Step 2: Attach the Disposable Tip
• Select a box containing the
correct size tips to be used.
• Place the pointed end of the
micropipette shaft into one of
the tips.
• Press down and twist slightly
to ensure an airtight seal, then
the
lift-upAttaching
the micropipette
with
disposable tip
tip attached.
•YOU MUST NEVER USE THE
MICROPIPETTE WITHOUT A
TIP ATTACHED !!!
Example of tip sizes:
Operating the Micropipette-3
Step 3: Depress the
Plunger to the First
Stop
Step 4: Immerse Tip in Sample
•To aspirate the sample
Step
Draw
thethe
sample
into 5:
the
tip, up
allow
pushbutton to return
slowly and smoothly to
the fully extended UP
POSITION. NEVER LET
THE PLUNGER SNAP UP!
Step 5: Pause
•Wait a few seconds to ensure that the full volume
of sample is drawn into the plastic tip. WAIT
LONGER FOR LARGER VOLUMES or MORE VISCOUS
("SYRUP-LIKE") SUBSTANCES.
Operating the Micropipette-4
Step 6: Withdraw the Tip
• Remove the tip from the sample liquid. Be
sure No liquid remain on the OUTSIDE of the
tip.
• Visually
examine the tip to make sure it
contains reagent and that there are no
bubbles present.
Operating the Micropipette-5
Step 7:
8: To
Dispense
thethe
Sample
•Step
dispense
solution from the pipette:
a) Touch the tip end to the side wall of the receiving
vessel.
b) Depress the plunger to the FIRST STOP.
c) Pause for at least one second .
d) Press the plunger to the SECOND STOP (the second
point, of greater resistance, at the bottom of the
stroke) to expel any residual liquid in the tip (like
"blowing out" a glass pipette).
(a) Start
Dispensing
(b) 1st
Stop =
Dispense
(c) 2nd
Stop =
Expel
Operating the Micropipette-6
Step 8: Withdraw the Pipette
•With the plunger fully depressed, withdraw the
pipette from the receiving vessel carefully.
Step 9: Release the Plunger
•Gently allow the plunger to return
to the UP position.
Step
•Step11:
10: Discard
Discard the
theTip
tip by
depressing the tip ejector button,
as shown below. A fresh tip should
be used for each sample to prevent
sample carryover.
Press ejector button to discard tip.
Step-wise Operation of the
Micropipette
•
•
•
•
•
•
Set volume.
Attach disposable tip.
Depress the plunger to 1st stop.
Immerse tip in sample and Draw up sample.
Withdraw the tip.
Dispense the sample by pushing the plunger
to the 2nd stop.
• Withdraw the pipette and release the
plunger.
• Discard the tip.
Pippetting Guidelines and Precautions
For optimal reproducibility, use the following pipetting
procedures:
(1) Consistent SPEED and SMOOTHNESS when you
press and release the PLUNGER
(2) Consistent pressure on the PLUNGER at the FIRST
STOP
(3) Consistent and sufficient IMMERSION DEPTH
(4) Nearly VERTICAL POSITIONING of pipette
(5) AVOID ALL AIR BUBBLES: Since the plastic pipette
shaft can be damaged if liquids are drawn beyond
the tip into the shaft.
(6) NEVER lay the pipette on its SIDE nor INVERT the
pipette if liquid is in the tip.
Part Ⅲ: Accuracy and Precision
• In Science, we want measurements to
be both accurate and precise.
• What is the difference between them?
Accuracy and Precision
• Accuracy is a measure of rightness.
– It means "capable of providing a correct reading or
measurement."
– It refers to how closely a measured value agrees
with the actual value.
• Precision is a measure of exactness.
– Precise means “repeatable, reliable, getting the
same measurement each time.”
– It refers to how closely individual measurements
agree with each other.
Can you hit the bull's-eye?
Three
targets with
three
arrows each
to shoot.
How do
they
compare?
Both
accurate
and precise
Precise
but not
accurate
Neither
accurate
nor precise
Error Sources
• Instrumental errors
• Natural errors
• Personal errors
Instrumental Errors
• Caused by imperfections in instrument
construction or adjustment
• Examples – imperfect spacing of
graduations, nominally perpendicular
axes not at exactly 90°, level bubbles
or crosshairs misadjusted …
• Fundamental principle – keep instrument
in adjustment to the extent feasible
Natural Errors
• Errors caused by conditions in the
environment that are not nominal
• Examples – temperature different from
standard, atmospheric pressure
variation, gravity variation, magnetic
fields, wind and so on
Personal Errors
• Errors due to limitations in human
senses or dexterity
• Examples – ability to read a micrometer,
steadiness of the hand, estimate
between graduations, …
• These factors may be influenced by
conditions such as weather, insects,
hazards, …
• Some of the afore-mentioned errors
(instrumental, natural, and personal) occur in a
systematic manner and others behave with
apparent randomness.
• They are therefore referred to as systematic and
random errors.
Systematic Errors
• Are TYPICALLY present.
• Measurements are given
as:
• Sources:
– Instrumental,
physical and
human limitations.
• Example: Device is
out-of calibration.
Measurement + Systematic Error
OR
Measurement - Systematic Error
• How to minimize
them?
– Careful calibration.
– Best possible
techniques.
Random Errors
• ALWAYS present.
• Measurements are often
shown as:
• Sources:
– Operator errors
– Changes in experimental
conditions
• How to minimize them?
– Take repeated
measurements and
calculate their average.
Measurement ± Random Error
(1) Accuracy and error
Absolute error : E = Xi - XT
Where, Xi: measured value; XT: True value
Relative error :
(2) Precision and deviation
standard deviation (SD or S)
Mean:
X=
x1  x2  xn
n
Relative Standard Deviation (RSD) or Coefficient of
Variation (CV)
S
CV   100%
X
Accuracy and Precision of Micropipette
Volume
P20
P100
P200
P1000
Accuracy
Precision
ul
error（ul）
error（％）
S.D.（ul）
R.S.D.（ul）
2
±0.1
±5.0
≤0.03
≤1.5
5
±0.1
±2.0
≤0.04
≤0.80
10
±0.1
±1.0
≤0.05
≤0.50
20
±0.2
±1.0
≤0.06
≤0.30
20
±0.35
±1.8
≤0.10
≤0.5
50
±0.40
±0.8
≤0.12
≤0.24
100
±0.80
±0.8
≤0.15
≤0.15
50
±0.5
±1.0
≤0.20
≤0.40
100
±0.8
±0.8
≤0.25
≤0.25
200
±1.6
±0.8
≤0.30
≤0.15
200
±3
±1.5
≤0.60
≤1.30
500
±4
±0.8
≤1.0
≤0.20
1000
±8
±0.8
≤1.5
≤0.15
•
•
•
How to use electronic analytical
balance
Operation
1. Check to ensure that the horizontal
position of the balance is level.
Each balance is equipped with a
level indicator.
2. Switch on /power
3. Tare ,make scale “0.000”
4. Weight：record the weight
Attention
Horizontal-level bubble gauge
PRACTICE
Pipette
1) Prepare a electronic balance
2) Prepare 200 ml of distilled water
3) Prepare a glass beaker
4) Weigh the glass beaker and Tare out the balance (i.e.,
make the scale 0.000)
5）Select glass pipettes and rubber bulb
6）Dispense 3.8ml (or 1.6ml) distilled water in the
beaker and read the balance and record the weight
7) Repeat 2~3 times and record the weight of each
time
8）Calculate the Volume using the recorded weight and
Temp-Density conversion table.
9） Calculate
Micropipette
1) Prepare a electronic chemical balance
2) Prepare 200 ml of distilled water and measure the
temperature
3) Prepare a glass beaker
4) Weigh the glass beaker and Tear out the balance (i.e., make
the scale 0.000)
5) Dispense 550 µl (or 160 µl) distilled water in the beaker and
read the balance and record the weight
6) Repeat 2~3 times and record the weight of each time.
7) Calculate the Volume using the recorded weight and TempDensity conversion table.
8) Calculate
Results
Micropipette
Pipette
Roll
number
Roll
number
number
550 (or 160 )
Required
volume
(µl)
3.8 (or 1.6)
Required
volume
(ml)
1 2
3
4 5 6 7 8 9 1
1
0 1
1
2
number
Measured
Mass (g)
Measured
Mass (g)
Measured
Volume
(ml)
Measured
Volume
(ml)
Absolute
Error
Absolute
Error
Relative
Error
Relative
Error
Mean of
measured
volume
Mean of
measured
volume
SD
SD
1 2
3
4
5
6
7
8
9
1
0
1
1
Volume (ml)= Mass (g) / Density (g/cm3). Note the significant digits.
1
2
Table. the different density of water at different temperature
T(0C)
Density(g/cm3) T(0C) Density(g/cm3) T(0C) Density(g/cm3)
0
0.999 868
12
0.999 525
24
0.997 326
1
0.999 927
13
0.999 404
25
0.997 074
2
0.999 968
14
0.999 271
26
0.996 813
3
0.999 992
15
0.999 126
27
0.996 542
4
1.000 000
16
0.998 970
28
0.996 262
5
0.999 992
17
0.998 802
29
0.995 973
6
0.999 968
18
0.998 623
30
0.995 676
7
0.999 929
19
0.998 433
31
0.995 369
8
0.999 876
20
0.998 232
32
0.995 054
9
0.999 869
21
0.998 021
33
0.994 731
10
0.999 728
22
0.997 799
34
0.994 399
11
0.999 632
23
0.997 567
35
0.994 059
Discussion
• How about the accuracy and precision of
your operation (pipette and
micropipette)?
• What are the precautions when you
operate the pipette and micropipette?
Next experiment
• Spectrophotometry & Plotting of
Calibration Curve