Lab 1
GENERAL INSTRUCTION
1. An observation notebook should be kept for laboratory trials. Mistakes should not be
removed; they should be marked by a single line. The notebook may be collected by the lab
manager at any time and always be updated
2. Index: An index contains the title of each conducted experiment and the page number should
be included.
You should write everything that you do in the laboratory in your observation notebook. The
notebook should be organized by experiment only and not as a daily log. Each new
experiment should be start on a new page. The top of the page contains title, date and page
number. The page number is important for indexing and referring to previous experiments.
Each experiment should include the following:
i.
Title/Purpose: Every experiment should have a descriptive title.
ii.
Background Information: This section should include any information that is
pertinent /relevant to the execution/implementation of the experiment or the
interpretation of the results.
iii.
Materials: This section should include all materials, i.e., solutions or equipment that
will be considered necessary for experiment. Composition of all buffers should be
included, unless they are standard or included in a kit. Biological reagents should be
identified by their original source, for example, concentration, source, purity, and/or
restriction map for nucleic acids; and base sequence, for oligonucleotides.
iv.
Procedure: Write procedure and flow chart before you perform each experiment and
understood all step before you do it.
 You should include everything you do, including all volumes and amounts;
many protocols are written for general use and must be adapted for a specific
application.
 Writing a procedure helps you to remember about it and identify steps that may
be unclear.
 Some procedures can be several pages long and include more information than
is necessary for a notebook. Therefore, it is fine laboratory trained to have
another notebook including techniques that you use on an ordinary basis.
 If an experiment is a repeat of an earlier experiment, not necessary writing each
step, but refer the earlier experiment by page or experiment number. If you do
any modifications, note the modifications and reasons.
 Flow charts and tables are sometimes helpful for experiments that have many
parts and an experiment includes a set of reactions with multiple variables
respectively.
V.
Results: This section should include all raw data, together with gel photographs,
printouts, colony counts, graphs, autoradiographs, etc. This section should also
include your analyzed data; for example, transformation efficiencies or calculations
of specific activities or enzyme activities.
VI.
Conclusions/Summary: This is one of the most vital parts. It should include
summary of all results and conclusions. Extension
LABORATORY SAFETY PROCEDURES
a) Chemicals: Many chemicals used in the laboratory are hazardous. All manufacturers of these
chemicals are requested by law to supply the user with relevant information on any hazards
together with their chemicals. This information is supplied in the form of Material Safety
Data Sheets, or MSDS. This information includes the chemical name, CAS#, health hazard
data, including first aid treatment, physical data, fire and explosion hazard data, reactivity
data, spill or leak procedures, and any special precautions needed when handling this
chemical. In addition, MSDS information can be accessed on the Web on the Biological
Sciences Home Page. You are strongly advised to make use of this information prior to using
a new chemical, and definitely in the case of any accidental exposure or spill. The following
chemicals are particularly noteworthy in molecular laboratory:
 Phenol—can cause severe burns
 Acrylamide—potential neurotoxin
 Ethidium bromide—carcinogen.
These chemicals are not harmful if used properly: always wear gloves when using potentially
hazardous chemicals, and never mouth-pipette them. If you accidentally spray any of these
chemicals on your skin, immediately rinse the area thoroughly with water and inform the lab
manager. The wastes have to discard in appropriate containers.
b) Ultraviolet Light: Exposure to ultraviolet (UV) light can cause acute eye irritation. as the
retina cannot detect UV light, you can have serious eye damage and not realize it until 30
minutes to 24 hours after exposure. Therefore, always wear appropriate eye protection when
using UV lamps.
c) Electricity: The voltages used for electrophoresis are sufficient to cause electrocution. Cover
the buffer tanks during electrophoresis. Always turn off the power supply and unplug the
leads before removing a gel.
d) Glassware. Glass and plasticware used for molecular biology must be scrupulously clean.
Glassware should be rinsed with distilled water and autoclaved or baked at 150°C for 1 hour.
For experiments with RNA, glassware and solutions are treated with diethylpyrocarbonateto
inhibit RNases, which can be resistant for autoclaving. Plasticware, such as pipettes and
culture tubes, is often supplied sterile. Tubes made of polypropylene are turbid and resistant
to many chemicals, like phenol and chloroform; polycarbonate or polystyrene tubes are clear
and not resistant to many chemicals. Micropipette tips and microfuge tubes should be
autoclaved before use.
e) General Housekeeping: All common areas should be kept free of dirty dishes, disordered
equipment and should be put appropriately. Since you have only a limited amount of space of
your own, it is to your advantage to keep that area clean. As you will use common services,
all solutions and everything accumulated in refrigerator, an incubator, etc., must be labeled.
In order to limit confusion, each person should use his initials or another unique designation
for labeling plates, etc. Unmarked material found in freezers, refrigerators or incubators may
be discarded. All times mark the backs of the plates with your initials, date, and relevant
experimental data, e.g., strain numbers.
Disposal of Buffers and Chemicals
I.
Any uncontaminated, solidified agar or agarose should be discarded in the trash, not in the
sink, and the bottles rinsed well.
II.
Any media that becomes contaminated should be promptly autoclaved before discarding it.
Petri dishes and other biological waste should be discarded in biohazard containers, which
will be autoclaved prior to disposal.
III.
Organic reagents, e.g., phenol, should be used in a fume hood and all organic waste should
be disposed of in a labeled container, not in the trash or the sink.
IV.
Ethidium bromide is a mutagenic substance that should be treated before disposal and
handled only with gloves. Ethidium bromide should be disposed off in a labeled container.
EQUIPMENTS
Keep the equipment in good working condition. Don't use anything (any instrument) unless you
have been instructed in its proper use. Report any malfunction immediately. Rinse out /clean all
centrifuge rotors after use, in particular if anything spills/tumbles/ falls. Please do not waste
supplies use only what you need. If the supply is running low, please notify the lab manager
before it is completely exhausted. Occasionally, it is necessary to borrow reagent or equipment
from another lab.
Micropipettors
Most of the experiments you will conduct in the laboratory will depend on your ability to
accurately measure volumes of solutions using micropipettors. The accuracy of your pipetting
can only be as accurate as your pipettor, and several steps should be taken to ensure that your
pipettes are accurate and maintained in good working order. Then they should check for
accuracy following the instructions given by the instructor. If they need to be recalibrated, do so.
Since the pipettors will use different pipette tips, make sure that the pipette tip you are using is
designed for your pipettor.
PH Meter
Biological functions are very sensitive to changes in pH and hence, buffers are used to stabilize
the pH. A pH meter is an instrument that measures the potential difference between a reference
electrode and a glass electrode, often combined into one combination electrode. The reference
electrode is often AgCl2. An accurate pH reading depends on standardization, the degree of
static charge, and temperature of the solution.
Operation of pH Meter
1. Expose hole on side of electrode by sliding the collar down. Make sure there is sufficient
electrode filling solution in the electrode (it should be up to the hole). If not, fill with
ROSS filling solution only (Do not use any filling solution containing silver (Ag).
2. Ensure that sample to be pHed is at room temperature and is stirring gently on the stir
plate.
3. Calibrate the pH meter with the two solutions that bracket the target pH - 4 and 7 or 7
and 10 as follows:
4. Press the CAL key to initialize the calibration sequence. The last calibration range will
be displayed (e.g. 7-4). Press YES to accept or use the scroll keys to select a different
range. Press YES to accept.
5. The number 7 will light up on the left hand side of the screen indicating that the meter is
ready to accept the pH 7 standard buffer. Rinse off electrode and place in fresh pH 7
standard buffer solution. The READY light will come on when the value has stabilized.
Press YES to accept the value.
6. The number 4 (or 10) will light up next indicating that the meter is ready to accept the
pH 4 (or 10) standard buffer solution. Rinse off electrode and place in fresh pH 4
standard buffer solution. The READY light will come on when the value has stabilized.
Press YES to accept the value.
7. SLP will be displayed. The meter will then go MEASURE mode.
8. Rinse electrode and place into sample. The READY light is displayed when signal is
stable.
Autoclave
Place all material to be autoclaved on an autoclavable tray. All items should have indicator tape.
Separate liquid from solid and autoclave. Make sure the lids on all bottles are loose. Make sure
the chamber pressure is at zero before opening the door.
Autoclave Operating Procedures
1. Place items to be autoclaved in the autoclave and close the door. Some autoclaves require
that you also lock the door after it's closed.
2. Set time - typically 20 minutes.
3. Temperature should be set at 121 deg C already, but double-check and change if
necessary.
4. Set cycle: If liquid, set "liquid cycle" or "slow exhaust". If dry, set "dry cycle" or "fast
exhaust" + dry time.
5. Start the cycle. On some autoclaves, the cycle starts automatically at step 5. On others,
turn to "sterilize".
6. At the end of the cycle, check that: a. the chamber pressure is at 0; b. the temp is <100
deg C
7. Open door.
8. Remove contents using gloves and immediately tighten all caps.
NanoDrop 8000 Spectrophotometer
NanoDrop 8000 Spectrophotometer is a full-spectrum (220-750nm) instrument that measures 8
individual 1 ul samples with high accuracy and reproducibility. The surface retention system
holds the sample in place eliminating the need for cumbersome cuvettes and other sample
containment devices. Clean-up is accomplished in seconds. In addition, the NanoDrop 8000 has
the capability to measure highly concentrated samples without dilution (50X higher
concentration than the samples measured by a standard cuvette spectrophotometer).
The NanoDrop 8000 has the capability to measure Nucleic acid concentration and purity of
nucleic acid samples up to 3700 ng/ul (dsDNA) without dilution. After Up to eight 1 ul samples
are pipetted onto the sample pedestal using a low volume multi-channel pipettor. Each position
is actually the end of a fiber optic cable (the receiving fibers). A second set of fiber optic cables
(the source fibers) are brought into contact with the liquid samples causing the liquid to bridge
the gaps between the fiber optic ends. The path lengths are automatically controlled to 1mm and
0.2 mm paths. Readings are acquired through sequential measurement across the 8 positions. A
pulsed xenon flash lamp provides the light source and a spectrometer utilizing a linear CCD
array is used to analyze the light that passes through the samples. The instrument is controlled by
PC based software, and the data is logged in an archive file on the PC.
Applications
 Nucleic acid concentration and purity of nucleic acid samples up to 3700 ng/ul
(dsDNA) without dilution
 Fluorescent dye labeling density of nucleic acid microarray samples
 Purified protein analysis (A280 nm) up to 100 mg/ml (BSA)
 Expanded spectrum measurement and quantization of fluorescent dye labeled
proteins, conjugates, and metalloproteinase
 Bradford Assay analysis of protein
 BCA Assay analysis of protein
 Lowry Assay analysis of protein
 Pierce Protein 660 nm analysis
 Cell density measurements
 General UV-V is spectrophotometry
Procedures
1. Plug in the ND and PC to the power supply
2. Double click the software “ND 8000‟‟ from the PC then „‟click on the listed molecule
that you are going to measure
3. Initialize the ND by 1.5-2µL of nuclease free water
4. click on „‟ load sample ID file‟‟ and select from the listed „‟ plate ID‟ that you are
interested
5. Click on „‟continue „‟ and please look the well color until it becomes „‟yellow‟‟
6. Click on „‟All active ON/OFF‟‟ option and write your sample ID on the given space and
please look the plate well are „‟Green color‟‟
7. Blank the ND by 1.5-2µL of the resuspended solvent of your sample
8. Dispense the samples onto the lower measurement pedestal ensuring the samples “touchoff” (contact) the lower pedestal. Carefully withdraw the pipettor before releasing the
pipette‟s dispensing mechanism. Visually verify all samples are correctly transferred to
their respective pedestals.
9. Close the sampling arm and initiate a spectral measurement using the operating software
on the PC. The sample columns are automatically drawn between the upper and lower
measurement pedestals and the spectral measurement made.
10. Click on „‟Measure‟‟ option which is located at the left corner of the software
11. Click on „‟show report‟‟ and „‟save as window‟‟ option which is located at the top of the
software to rescored your data
12. When the measurement is complete, open the sampling arm and wipe the samples from
both the upper and lower pedestals using a soft laboratory wipe.
NOTE: - Wiping the sample from both the upper and lower pedestals upon completion of each
sample measurement is usually sufficient to prevent sample carryover and avoid residue buildup