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Laboratory Safety Procedures for Molecular biology lab.
A. GENERAL
1. Work carefully and cautiously in the laboratory, using common sense and good
judgment at all times.
2. EATING AND DRINKING ARE PROHIBITED in the laboratory.
3. Long hair must be tied back during laboratory sessions.
4. Keep sinks free of paper or any debris that could interfere with drainage.
5. Lab tables must be clear of all items that are not necessary for the lab exercise.
6. Wash hands and the lab tables with the appropriate cleaning agents before and after
every laboratory session.
B. SHARP OBJECTS AND BROKEN GLASS
1. Pointed dissection probes, scalpels, razor blades, scissors, and microtome knives must
be used with great care, and placed in a safe position when not in use.
2. Containers designated for the disposal of sharps (scalpel blades, razor blades,
needles; dissection pins, etc.) and containers designated for broken glass are present in
each laboratory. Never dispose of any sharp object in the regular trash containers.
3. Do not touch broken glass with bare hands. Put on gloves and use a broom and
dustpan to clean up glass. Dispose of ALL broken glass in the specific container marked
for glass. Do not place broken glass in the regular trash.
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C. INSTRUMENTS AND EQUIPMENT
Care must be used when handling any equipment in the laboratory. Students are
responsible for being familiar with and following correct safety practices for all
instruments and equipment used in the laboratory.
G. BODY FLUIDS
Special precautions are to be followed in all laboratories using any body fluids, such as
blood, saliva, and urine, because of the potential to transmit disease-causing organisms.
1. Use gloves in all laboratory experiments that involve the use of body fluids.
2. All contaminated material, such as slides, coverslips, toothpicks, lancets, alcohol
swabs, etc., must be placed in a biohazard bag for proper disposal and should never be
reused.
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Blood Collection Tubes:
1.EDTA TUBES:
EDTA tube is widely used for clinical hematology as well as various kinds of blood cell
test instrument. It makes use of EDTA as anticoagulant. Meanwhile, it offers a
comprehensive protection for blood cell, especially for protecting the blood platelet, so
that it can effectively stop the gathering of blood platelet and makes the form and
volume of blood cell uninfluenced in a long time. EDTA tube can be used for DNA
extraction and plasma isolation.
2.NO ADDITIVE TUBES:
No additive tube is used for blood collection and storage for biochemistry, immunology
and serology tests in medical inspection. It can provide enough and non-polluted serum
specimen for clinical tests.
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- Isolation of Serum and Plasma
Note:
Blood Plasma contains many vital proteins including fibrinogen, globulins and human
serum albumin.
Serum refers to blood plasma in which clotting factors (such as fibrin) have been
removed naturally by allowing the blood to clot prior to isolating the liquid component
1- Isolation of Serum from Blood.
1. Collect the blood in Red top tubes and allow it to coagulate for 30-60 minutes.
2. Centrifuge at 3000 rpm for 15 minutes.
3. Transfer the serum to 1.5 ml tubes.
2- Isolation of Plasma from Blood.
1. Collect the blood in EDTA (purple top) tubes.
2. Centrifuge at 3000 rpm for 15 minutes.
3. Transfer the Plasma to 1.5 ml tubes.
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DNA EXTRACTION
Isolation of DNA from Whole Blood using Qiagen DNA Purification Kit.
Principle
The Qiagen DNA Purification Kit relies on biological or environmental specimens as a
source of genomic, mitochondrial or viral DNA. Typical sample types include: whole
blood, buffy coat, cultured cells, body fluids, animal or plant tissue, and microbes.
Most mammalian whole blood and bone marrow specimens contain both nonnucleated cells (red blood cells) and nucleated cells (white blood cells) which contain
DNA. When purifying DNA from these whole blood or bone marrow specimens, the red
blood cells, which lack genomic DNA, are first lysed to facilitate their separation from
the white blood cells. DNA is purified from cells such as white blood cells, animal tissue,
cells contained in body fluids, or microbes, by first lysing the cells with an anionic
detergent in the presence of a DNA stabilizer. The DNA stabilizer works by limiting the
activity of DNases that are contained in the cell and elsewhere in the environment.
Contaminants, such as proteins, are removed by salt precipitation. Finally, the genomic
DNA is recovered by precipitation with alcohol and dissolved in a buffered solution
containing a DNA stabilizer.
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DNA purification from 5 ml whole blood
RBC Lysis
1)
Add 5 ml of whole blood into a 50 ml centrifuge tube containing 15 ml
RBC lysis solution. Invert to mix and incubate 10 minutes at room
temperature. The tube will be inverted again at least 20 times during the
incubation.
2)
Centrifuge the tube at 3,000 RPM for 15 minutes and remove the
supernatant (leaving behind the white cell pellet and about 200µl of the
residual liquid).
3)
vortex the tube vigorously to re-suspend the cells in the residual liquid.
This will be greatly facilitated cell lysis in Step 4 below.
Cell Lysis
4)
Add 5 ml Cell Lysis solution to the re-suspended cells and pipette up and
down to lyse the cells. If cell clumps are visible, vortex the tube for 40
seconds and incubate the sample at 37°C until the solution is
homogeneous. Samples are stable in cell lysis solution for at least 18
months at room temperature.
Protein Precipitation
1)
Add 1.7 ml Protein precipitation solution to the cell lysate.
2)
Vortex the sample at high speed for 50 seconds to mix the protein precipitation
solution uniformly with the cell lysate.
3)
centrifuge the sample at 3,000 RPM for 15 minutes. The precipitated proteins
should form a tight, dark brown pellet.
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DNA Precipitation
1) pour the supernatant containing the DNA into a clean 50 ml centrifuge tube
containing 5 ml 100% Isopropanol (2-propanol).
2)
Mix the sample by inverting gently 20 times until the white threads of DNA
formed a visible clump.
3) centrifuge the sample at 3,000 RPM for 3 minutes. The DNA will visible as a small
white pellet.
4) poured off the supernatant and drain tube briefly on clean absorbent paper. Add
5ml 70% ethanol and invert the tube several times to wash the DNA pellet.
5) centrifuge the sample at 3,000 RPM for 1 minute. Carefully pour off the ethanol.
6)
invert the tube and drain on clean absorbent paper and allow to air dry 10-20
minutes.
DNA Hydration
1)
Add 100-350 µl DNA hydration solution.
2)
Rehydrated
DNA by incubating at 65°C for 1 hour and overnight at room
temperature. Tape tube periodically to aid in dispersing the DNA.
3)
Centrifuged the sample briefly and then transferred to a 1.5 ml microfuge tube.
4)
The purity and concentration will be checked for each DNA sample.
5)
From each sample 100 µl working DNA (50ng/ µl) will be labeled and stored at 4°C
or – 20° C.
6)
Original stock of each sample of DNA will be labeled and stored at -80°C.
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