training report
TRANSCRIPT
Rajiv Gandhi Centre for Biotechnology
Department of Biotechnology, Ministry of Science & Technology, Thiruvananthapuram, Kerala
SNP Genotyping and Cell Culture Techniques
Human Molecular Genetics Lab
Submitted by
Stevin Wilson
Aruna Mohan
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Acknowledgement
We would like to express our gratitude to all those who gave us the possibility to
complete this project.
First of all we would like to thank Dr.Radhakrishnan Pillai, Director, Rajiv Gandhi Centre
for Biotechnology and Research for giving us the opportunity to be a part of the institute.
Our heartfelt thanks to Dr. Moinak Banerjee for letting us use his lab, for his outstanding
support and knowledge he shared with us.
This report would not have been possible without the help of Ms. Swathy B, Mr.Sanish
Sathyan, Ms. Sarada Lakshmi,Mr. Shabeesh Balan, Mr. Antony KP and Ms. Anaswara
Ashok. They steered us through the basics of all techniques practiced in the lab. We
would like to thank Mrs. Veluthai for providing us facilities in the lab.
We also appreciate the support and encouragement of our fellow lab mates during the
tenure.
Above all we thank the Almighty for His blessings without which this training would have
been just a dream.
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Contents
Section-A: SNP Genotyping
A1.DNA ISOLATION 6
A2.DNA QUANTIFICATION AND QUALITY CHECK OF DNA 10
A3.POLYMERASE CHAIN REACTION 13
A4.AGAROSE GEL ELECTROPHORESIS 19
A5.DNA SEQUENCING 21
A6.RESTRICTION FRAGMENT LENGTH POLYMORPHISM 27
TABLES AND FIGURES 30
Section-B: Cell Culturing B1.PREPARING AN ASEPTIC ENVIRONMENT 34
B2.PREPARATION OF CELL GROWTH MEDIUM AND CULTURE CONDITIONS 36
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B3. CHECKING CELLS 39
B4.SUBCULTURE FROM ADHERENT CULTURED CELLS 40
B5.COUNTING CELLS USING HEMOCYTOMETER 42
B6.FREEZING CELLS 43
B7.CELL REVIVAL 45
B8.CELL SEEDING FOR DNA/RNA ISOLATION 47
B9.DRUG TREATMENT TO CELL LINE 48
B10.DNA ISOLATION FROM CULTURED CELL LINES 49
B11. QUANTIFICATION USING NANODROP™ 50
B12.RNA ISOLATION FROM CULTURED CELL LINES 51
B13. ASSESSMENT OF RNA QUALITY AND QUANTITY 52
B14.cDNA PREPARATION 53
B15. REAL-‐TIME PCR 55
REFERENCES 58
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Section-A
SNP GENOTYPING- BASIC TECHNIQUES
6
Experiment -A1
DNA ISOLATION Before any type of analysis can be performed, DNA must be isolated.
The success of all subsequent procedures depends on the availability of
sufficient amounts of DNA of the appropriate quality. Many different methods
and technologies are available for the isolation of genomic DNA. All methods
involve disruption and lysis of the starting material followed by the removal of
proteins and other contaminants and finally recovery of the DNA.
The method used here is “Phenol –chloroform DNA extraction’’ from blood
sample. This method was found by was originally devised by Piotr Chomczynski
and Nicoletta Sacchi and published in 1987 (referred to as Guanidinium
thiocyanate-phenol-chloroform extraction).
Principle:
Human blood consists of enucleated RBCs and nucleated WBCs and
platelets. The blood is first suspended in RBC lysis buffer. This buffer consists
of chloride ions, which will enter the cell as a result of which the cell enlarges,
the membrane integrity thereby weakens and RBC gets lysed subsequently.
EDTA is a chelating agent for metal ions, which are cofactors for nucleases
thus inhibiting their activity. The pellet obtained after centrifugation consists
mainly of nucleated cells: WBCs and platelets. In order for the cell to be lysed,
the lipid walls must be broken down. Cell walls, cell membranes, and nuclear
membranes are also broken down by the action of the blender. Incubation at
56⁰c in the presence of Proteinase K and SDS is used to partially digest
cellular proteins and loosen the association between proteins and chromosomal
DNA and to degrade cellular RNA.The cell lysate is then treated with buffer
saturated phenol: chloroform. The DNA remains in the aqueous phase while
the cellular proteins are extracted into the organic phase. The aqueous phase
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is often extracted a second time with phenol: chloroform to ensure complete
removal of the proteins. The aqueous phase, containing the DNA, is then
washed with ethanol and then dissolved in water or TE Buffer. DNA extracted
this way is generally high molecular weight and double stranded is therefore
suitable for either RFLP analysis or PCR amplification.
Reagents required:
• 20% SDS (sodium dodecyl sulphate)
• Absolute ethanol
• EDTA (0.5M)-pH8.0
• NaCl (5M)
• Tris saturated phenol- pH-8.0
• Chloroform –isoamyl alcohol (24:1)
• Sodium acetate (3M)
• 70% ethanol
• Proteinase K -20mg/ml
Buffers used:
• RBC lysis buffer (Tris: EDTA: NaCl-30: 5:50mM)
• WBC lysis buffer (NaCl: EDTA-75: 2mM)
• T.E buffer (Tris: EDTA10: 1mM)
Preparation of buffer solution:
RBC lysis buffer (TEN-30: 5:50mM)
− 1 M Tris - 7.5 ml
− 0.5M EDTA-2.5 ml
− 5M NaCl – 2.5 ml
− Distilled water-250 ml
WBC lysis buffer: (N: E-75: 2mM)
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− 0.5M EDTA – 1 ml
− 5 M NaCl - 3.75 ml
− Distilled water - 250 ml
TE buffer: (10:1mM)
− 1M Tris – 1 ml
− 0.5M EDTA - 0.2 ml
− Distilled water – 100 ml
Equipment and instruments:
o Deep freezer (-80°C)
o Water bath
o Centrifuge (Rota 4R)
o Micro centrifuge (Hitachi Himac CR21E)
o Laminar air flow chamber
o Incubator
o Homogenizer
o Pipettes
Procedure
Day 1
• Intravenous blood specimen collected in EDTA tubes can be stored for a
longer period of time at -20⁰c or -80⁰c for later extraction of DNA.
• In such specimens equal volume of RBC Lysis buffer is added.
Day2
• Remove the blood samples from freezer and thaw it in a water bath for
10-15 minutes.
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• Centrifuge the tubes at 10,000rpm for 10minutes at 15⁰c. After the
centrifugation carefully remove the supernatant without disturbing the
pellet.
• Add equal volume WBC lysis buffer to the pellet and dissolve the pellet
thoroughly. Then add Proteinase k of 100µ /ml and SDS to make 2%
concentration in the final volume. Mix well and incubate the samples at
37 ⁰c overnight in a water bath.
Day3
• When the cell are fully digested, take out the lysate add equal volume of
Tris –saturated phenol (pH8.0).
• Centrifuge for 10minutes at 10,000rpm at 4⁰c.
• Collect the supernatant into a fresh tube and add 5ml of Tris Phenol. Mix
the contents of the tube gently for 2minutes and then add 5ml of
Chloroform+ isoamyl alcohol (25:24:1). Mix well and centrifuge at 10,000
rpm for 10minutes.
• Transfer the upper aqueous layer carefully into another centrifuge tube.
• Add equal volumes of Chloroform+ isoamyl alcohol (24:1) to the
supernatant and mix gently for a minute and centrifuge at 10,000rpm for
10minutes.
• Transfer the aqueous phase to a fresh tube.
• Add 1/10th the volume of 3M sodium acetate and equal volumes of
chilled absolute alcohol, mix gently to precipitate DNA.
• Spool out the DNA lump in a fresh 1.5ml tube and decant the alcohol.
• Wash the DNA twice with 70% alcohol.
• Dry the pellet and ensure that whole alcohol is dried off.
• Dissolve the pellet in TE buffer.
• Store the DNA samples at 4⁰c or -20⁰c or-80⁰c for future use.
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Experiment-A2
DNA QUANTITATION AND QUALITY CHECK OF DNA
Prior to any analysis, DNA samples should be quantitated and checked
for purity of DNA. The amount of light that a sample absorbs at a particular
wavelength is measured and used to determine the concentration of the sample by
comparison with appropriate standards or reference data. The most commonly
used methodologies for quantifying the amount of nucleic acid in a preparation
are: (i) gel electrophoresis and (ii) spectrophotometric analysis. Here the sample
amount being less, the latter method is preferred.
a) Spectrophotometric Determination
The spectrophotometric quantification of DNA is based on Beer-Lambert’s law that
gives the linear relationship between absorbance and concentration of absorbing
species:
A= λ × b × c
Where A is measured absorbance, λ is wavelength dependent absorptivity
coefficient, b is path length and c is analyte concentration.
Analysis of UV absorption by the nucleotides provides a simple and accurate
estimation of the concentration of nucleic acids in a sample. Purines and
pyramidines in nucleic acid show absorption maxima around 260nm (e.g. dATP :
259nm ; dCTP : 272nm ; dTTP : 247nm) if the DNA sample is pure without
significant contamination from proteins or organic solvents. The ratio of
OD260/OD280 genomic DNA should be determined to assess the purity of the
sample. This method is however limited by the quantity of DNA and the purity of
the preparation. Accurate analysis of the DNA preparation may be impeded by the
presence of impurities in the sample or if the amount of DNA is too little. In the
estimation of total genomic DNA, for example, the presence of RNA, sheared DNA
etc. could interfere with the accurate estimation of total high molecular weight.
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Based on its structure, DNA absorbs light in the ultraviolet range, specifically
at a wavelength of 260nm. A value of 1 at OD₂₆₀ is equal to 50ng/µl double-
stranded DNA, therefore to calculate the concentration of DNA ; the following
formula can be used:
Concentration DNA =260nmabs × 50ng/ µl
Purity of DNA sample can also be calculated based upon its absorbance of
light. A pure sample of DNA has a 260nm/280nm ratio of 1.8. Ratios deviating
from this usually indicate contamination of the sample with proteins, organic
solvents or RNA or could indicate degradation of the DNA sample.
Procedure
Determination of DNA Concentration by Spectrophotometry:
• Take 2 µl of DNA preparation and dilute it to 100 µl with Double Distilled
water and mix well.
• The spectrophotometer is calibrated at 260nm and 280nm with 100 µl
Double Distilled water.
• Measure OD of the diluted DNA aliquot at 260nm and 280nm using
cuvette. Calculate the OD260/OD280 ratio.
Quality Assessment:
A ratio of OD values at 260nm and 280nm indicates the purity of the extracted
DNA sample. If the ratio is within 1.6 to 2 range, the DNA is considered as
clear and free from contaminants.
An OD ratio less than 1.6 indicate the residual proteins or phenol
contamination, whereas ratio of more than 2.0 indicates residual RNA
contamination.
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Quantity Assessment:
If the OD value at 260nm of extracted sample is 1.00, then the concentration of
DNA is 50µg/ml.
So DNA concentration of the extracted sample = OD at 260nm × 50 × Dilution
factor
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Experiment-A3
POLYMERASE CHAIN REACTION (PCR)
The polymerase chain reaction is a technique widely used in molecular biology,
microbiology, genetics, diagnostics, clinical laboratories, forensic science,
environmental science, hereditary studies, paternity testing, and many other
applications. The name, polymerase chain reaction, comes from the DNA
polymerase used to amplify a piece of DNA by in vitro enzymatic replication.
The DNA polymerase enzyme, thus doubling the number of DNA molecules,
replicates the original molecule or molecules of DNA. Then each of these
molecules is replicated in a second "cycle" of replication, resulting in four times
the number of the original molecules. Again, each of these molecules is
replicated in a third cycle of replication. This process is known as a "chain
reaction" in which the original DNA template is exponentially amplified. With
PCR it is possible to amplify a single piece of DNA, or a very small number of
pieces of DNA, over many cycles, generating millions of copies of the original
DNA molecule. PCR has been extensively modified to perform a wide array of
genetic manipulations, diagnostic tests, and for many other uses.
Applications:
The polymerase chain reaction is used by a wide spectrum of scientists in an
ever-increasing range of scientific disciplines. In microbiology and molecular
biology, for example, PCR is used in research laboratories in DNA cloning
procedures, Southern blotting, DNA sequencing, recombinant DNA technology,
to name but a few. In clinical microbiology laboratories PCR is invaluable for
the diagnosis of microbial infections and epidemiological studies. PCR is also
used in forensics laboratories and is especially useful because only a tiny
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amount of original DNA is required, for example, sufficient DNA can be
obtained from a droplet of blood or a single hair.
Principle
The polymerase chain reaction (PCR) is a method for oligonucleotide primer
directed enzymatic amplification of a specific DNA sequence of interest. This
technique is capable of amplifying a sequence 105 to 106-fold from nanogram
amounts of template DNA within a large background of irrelevant sequences
(e.g. from total genomic DNA). A prerequisite for amplifying a sequence using
PCR is to have known, unique sequences flanking the segment of DNA to be
amplified so that specific oligonucleotides can be obtained. It is not necessary
to know anything about the intervening sequence between the primers. The
PCR product is amplified from the DNA template using a heat-stable DNA
polymerase from Thermus aquaticus (Taq DNA polymerase) and using an
automated thermal cycler to put the reaction through 30 or more cycles of
denaturing, annealing of primers, and polymerization.
After amplification by PCR, the products are separated by polyacrylamide gel
electrophoresis and are directly visualized after staining with ethidium
bromide. Ethidium bromide is added to the agarose to stain the DNA. Ethidium
bromide, a fluorescent dye binds tightly to the DNA double helix and glows
when illuminated with ultraviolet light. This lets us see where the separated
DNA fragments end up.
Primer Design Online tools
NCBI database - reference, database of SNP, Genes etc.
UCSC – Insilco PCR
Primer 3 – to design primer
Gene pipe – Alternative for above
1. Get the required SNP and flanking sequence from ncbi database
2. Copy and Past the sequence at Primer3 giving required parameter values.
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3. We obtain a primer design.
4. Verify the obtained primer by entering into UCSC.
5. Input the obtained primer into Primer Premier to check if it forms hairpin
structure.
Gradient PCR
The selection of the annealing temperature is possibly the most critical
component for optimizing the specificity of a PCR reaction. In most cases, this
temperature must be empirically tested. The PCR is normally started at 5°C
below the calculated temperature of the primer melting point (Tm). However,
the possible formation of unspecific secondary bands shows that the optimum
temperature is often much higher than the calculated temperature (>12°C).
Further PCR reactions with gradually increasing temperatures are required
until the most stringent conditions have been found. When a standard PCR
cycler is used, this method is the most time-intensive optimization strategy.
During the PCR, a temperature gradient, which can be programmed between
say 50 and 64°C, is built up across the thermo block. This allows the most
stringent parameters for every primer set to be calculated with the aid of only
one single PCR reaction.
The following reaction mixture was prepared for a required number of
reactions:
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COMPONENTS VOLUME(µl)
DNA
10X Buffer
dNTPs
20 pM forward primer
20 pM reverse primer
Taq polymerase (3U)
Sterile water
1
1
1
0.1
0.1
0.12
6.68
Total 10
Vortex the mixture briefly, then centrifuge at low speed. The Gradient PCR was
performed with the above tubes in a thermal cycle according to the following
protocol
Initial denaturation
Denaturation
Annealing
Extension
Final extension
Hold
94°C
94°C
550C to 65oC
72°C
72°C
4oC
5 minutes
30seconds
30seconds
30seconds
5 minutes
Forever
Protocol for a PCR reaction
1. The experiment has to be planned prior to any addition of reagents
(Number of primer pairs to be used, number of DNA templates, etc.).
Reagents Quantity per PCR tube in µl
Distilled water 6.68
10x Buffer 1.0
2.5µM dntp 1.0
20pM Forward Primer 0.1
35Cycles
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2. After doing so, make the appropriate cocktail/s and ensure complete
mixing by tapping the tube and quick spinning.
(N.B. Caution should be used to avoid contamination of reactions with even
small amounts of DNA. In addition, care should be taken to avoid
contamination of pipette with carryover amplification products from
previous reactions)
3. Pipette 9.3 µl of the appropriate cocktail directly into the bottom of a
sterile microeppendorf tube for each reaction. The tubes should be
labeled.
4. Add 0.7 µl of the DNA directly into the drop of cocktail in each tube and
ensure adequate mixing. Quick spin to collect the reaction mixture in the
bottom of the tube.
5. Place the tightly capped tubes in the temperature block and make sure
each is firmly seated by pressing on the tubes individually.
The PCR machine must now be programmed for the specific reaction
conditions desired. Each cycle in the polymerase chain reaction involves
three steps (denaturing, primer annealing, polymerization), and the
products are amplified by performing many cycles one after the other
with the help of the automated thermal cycler.
The Taq polymerase is heat stable, and remains active despite the high
denaturing temperature of each cycle. A representative set of reaction
conditions for 25-35 cycles is:
20pM Reverse Primer 0.1
5U/µl Taq Polymerase(NEB) 0.12
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Initial denaturation
Denaturation
Annealing
Extension
Final extension
Hold
94°C
94°C
550C
72°C
72°C
4oC
5 minutes
30seconds
30seconds
30seconds
5 minutes
Forever
6. After completion of the PCR reaction, remove the tubes from the
temperature block and place them in an eppendorf rack.
7. The reaction products are conveniently separated according to size by
electrophoresis through a 1% polyacrylamide gel at 75 V for 30-
45minutes, and visualized after staining the gel with ethidium bromide.
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Experiment-A4
GEL ELECTROPHORESIS
It is used to separate DNA fragments. Electrophoresis uses an electric current
to separate different-sized molecules in a porous, sponge-like matrix. Smaller
molecules move more easily through the gel pores than larger molecules.
The technique uses an agarose gel, made from highly purified seaweed. This
could be used to separate DNA molecules ranging from several hundred
nucleotides in length to over 10,000 nucleotides.
Materials required
∗ Horizontal gel electrophoresis
∗ Gel tray
∗ Gel combs
∗ Power supply unit
∗ Micro wave oven
∗ UV transilluminator
Reagents required
∗ 1X TAE buffer
∗ EtBr ( Ethidium Bromide)
∗ Gel loading dye(orange –G)
∗ Agarose (ultra pure)
Procedure
1. The gel is prepared by melting 0.3g of agarose in 30ml of 1X TAE buffer.
Add 2 µl of Ethidium bromide into the mixture and the mix was poured
into the gel tray taped on all sides.
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2. The combs are placed in the slots and the gel is ready to be used, once it
sets.
3. The tape is removed and the gel is submerged in a tank filled with 1XTAE
buffer that conducts electricity.
4. Using a pipette, DNA samples are loaded into the wells made in the
agarose gel. The DNA samples are colourless, but a blue tracking dye is
added to track the DNA migration through the gel.
5. The phosphate groups in the DNA backbone carry negatively charged
oxygen giving a DNA molecule an overall negative charge. In a n electric
current, the negatively charged DNA moves toward the positive pole of
the electrophoresis chamber.
6. The DNA molecules move through the gel by “reputation”- a reptile-like
snaking action through the pores of the agarose matrix. Smaller DNA
fragments migrate faster and further over a given period of time than do
larger fragments. This is how DNA fragments can be separated by size in
a agarose gel.
7. A photo of the gel is taken for later analysis.
8. The size of any DNA fragment can be determined by comparing it to
“markers”- DNA fragments of known sizes.
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Experiment-A5
DNA SEQUENCING
Principle
The principles of DNA replication were used by Sanger et al. (1974) in the
development of the process now known as Sanger dideoxy sequencing. This
process takes advantage of the ability of DNA polymerase to incorporate 2′, 3′-
dideoxynucleotides, nucleotide base analogs that lack the 3,′-hydroxyl group
essential in phosphodiester bond formation. Sanger dideoxy sequencing
requires a DNA template, a sequencing primer, DNA polymerase, nucleotides
(dNTPs), dideoxynucleotides (ddNTPs), and reaction buffer.
Four separate reactions are set up, each containing radioactively labeled
nucleotides and either ddA, ddC, ddG, or ddT. The annealing, labeling, and
termination steps are performed on separate heat blocks. DNA synthesis is
performed at 37 °C, the temperature at which the T7 DNA polymerase used has
the optimal enzyme activity.
DNA polymerase adds either a deoxynucleotide or the corresponding 2′, 3′-
dideoxynucleotide at each step of chain extension. Whether a deoxynucleotide
or a dideoxynucleotide is added depends on the relative concentration of both
molecules. When a deoxynucleotide (A, C, G, or T) is added to the 3′ end, chain
extension can continue. However, when a dideoxynucleotide (ddA, ddC, ddG, or
ddT) is added to
the 3´ end, chain extension terminates . Sanger dideoxy sequencing results in
the formation of extension products of various lengths terminated with
dideoxynucleotides at the 3′ end.
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DNA Template Preparation:
*PCR Strategies: Because cycle sequencing involves many cycles of template
denaturation and extension, adequate signal is produced in the sequencing
reaction. In selecting the strategy for generating PCR DNA templates to be used
for cycle sequencing, considering specificity and yield.
*Primer design and quantitation:
When you perform dye terminator cycle sequencing reactions on PCR template,
the
primer sequence, primer synthesis method, and primer purification method
can
greatly affect the quality of the sequencing data.
*Optimizing Primer Design:
• Primers should be at least 18 bases long to ensure good hybridization and to
minimize the probability of hybridizing to a second site on the target DNA.
• Use the recommended thermal cycling conditions for cycle sequencing,
because primers with Tm>45 °C produce better results than primers with lower
Tm.
• Avoid runs of an identical nucleotide, especially runs of four or more Gs.
• Avoid designing primers over a SNP. Consult SNP databases (dbSNP, SNP500,
and/or SNPbrowser™) for SNP locations.
• Keep the G-C content in the range 30 to 80%, preferably 50 to 55%. For
primers with G-C content less than 50%, you may need to increase the primer
length beyond 18 bases to maintain a Tm>45 °C.
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• Avoid primers that can hybridize to form dimers.
• Avoid palindromes because they can form secondary structures.
• The primer should be as pure as possible, preferably purified by HPLC.
PCR Contaminants That Affect Cycle Sequencing:
• Excess PCR primers – Compete with the sequencing primer for binding sites
and reagents in the sequencing reaction. Additional primers in sequencing
reactions using dye terminators result in the creation of multiple dye-labeled
sequence ladders and noisy data.
• Excess dNTPs – Can affect the dNTP/ddNTP balance of the sequencing reaction,
resulting in a decreased amount of short extension products.
• Nonspecific PCR products – Include primer-dimer artifacts and secondary PCR
products. Nonspecific PCR products behave as templates in the sequencing
reaction and cause the generation of multiple dye-labeled sequence ladders,
which result in noisy data. Any significant quantity of nonspecific PCR products
can result in poor quality sequencing data.
Procedure:
Following three steps are need for sequencing
a) Sequencing PCR
b) Sequencing clean up
c) Analysis using Genetic analyzer (Applied Biosystems 3730xl)
a) Sequencing PCR
For sequencing PCR, following constituent are needed, DNA(amplified product
from the primary PCR),Primer (Forward or reverse),Sequence mix ,Sequence
buffer and Water
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The following cocktail reaction mixture was prepared for the required
number of reactions.
Components Volume
DNA (PCR product) 1 µl
Sequencing Buffer (5X) 2µl
Sequence mix 0.25µl
Primer (forward) 0.4µl
Sterile water 6.35
Total 10µl
Sequencing PCR reaction mixture
i. Prepare sufficient sequencing master mix.
ii. Vortex and centrifuge the master mix briefly.
iii. Add 9.5µl of master mix to 0.5µl of cleaned PCR product.
iv. Place the samples on the PCR thermocycler using the following
conditions:
The Sequencing PCR is carried out as in condition shown below
PCR steps TEMPERATURE TIME
Denaturation
Extension
Hold
94C(35 cycles)
60°C
4°C
10 sec
4 min
Forever
b) Sequencing Reaction Clean Up:
Sequencing clean up is mainly done to purify the single stranded or double
stranded DNA product from primers, nucleotides, polymerases, oil and salt,
25
dNTPs, enzymes, short, failed PCR product so that they do not interfere with
the downstream application such as cloning sequencing or labeling.
Materials and reagents:
• PCR product
• Distilled water
• 125mM EDTA
• 3 M Sodium acetate
• Absolute ethanol
• 75% ethanol
• Formamide
Procedure:
1. Add 10µl water and 2 µl of 125mM EDTA to each sample and mix.
2. Add 2µl 3M sodium acetate (pH 5.2) and 50 µl 100% ethanol to each
sample. Incubate for 15minutes at room temperature.
3. Centrifuge for 12,000rpm for 20minutes at 26⁰c.
4. Decant the supernatant, add 100 µl 70% ethanol and centrifuge at
12,000rpm for 10 minutes at 26⁰c.Repeat the step for once more.
5. Decant the supernatant and air-dry the pellet at room temperature.
6. Add 10µl formamide to each DNA pellet and seal the plate.
7. Denature samples by heating to 96⁰c for 3minutes in the thermocycler
and immediately place on ice.
8. Prepare sample sheet and create a plate record on the analyzer.
9. Place the plate into a cassette and load on to the analyzer and run the
sequencing analysis.
c) Analysis using Genetic analyzer (Applied Biosystems 3730xl)
Instrumentation
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AB1 prism 3730 Genetic analyzer is an multi capillary automated system to
sequence, size and quantitate nucleic acids using multicolour fluorescent
labeling technology .AB1 PRISM Genetic analyzer software provides the
sequence data in the form of a chromatogram, where each nucleotide is
represented as a peak; C is represented by a blue peak, A by green, G by black
and T by red.
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Experiment -A6
Restriction Fragment Length Polymorphism
Restriction Fragment Length Polymorphism (RFLP) is a difference in
homologous DNA sequences that can be detected by the presence of fragments
of different lengths after digestion of the DNA samples in question with
specific restriction endonucleases. RFLP, as a molecular marker, is specific to a
single clone/restriction enzyme combination. RFLP is one technique used by
forensic scientists inDNA fingerprinting. It is also used for tracing ancestry,
studying evolution and migration of wildlife, and detection and diagnosis of
certain diseases. Most RFLP markers are co-dominant (both alleles in
heterozygous sample will be detected) and highly locus-specific.
The RFLP probes are frequently used in genome mapping and in variation
analysis (genotyping, forensics, paternity tests, hereditary disease diagnostics,
etc.).
RFLP methodology involves cutting a particular region of DNA with known
variability, with restriction enzymes, then separating the DNA fragments
by agarose gel electrophoresis and determining the number of fragments and
relative sizes. The pattern of fragment sizes will differ for each individual tested.
Materials required
DNA sample
Enzyme buffer
Restriction enzyme
Sterile distilled water
Instrumentation
Water bath
Agarose gel electrophoresis
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Digestion tubes
Micro centrifuge
Vortex mixer
Procedure
Restriction enzymes were selected using the software NEB cutter V2.0
(http://tools.neb.com/NEBcutter2/). This tool will take a DNA sequence
and find restriction enzymes that cut the sequence. Restriction enzymes
that cut the region of our SNP differentially based on the allele are
selected.rs2250889 was genotyped using restriction enzyme BsrBI.
Master Mix is prepared in an eppendorf tube for the required number of
reactions.
Reagents Quantity
Sterile distilled water 1.25µl
Buffer (NEB 2) 1.0µl
Enzyme 0.25µl
1. This mixer was first vortexed and spinned down using a micro centrifuge
2. The digestion tubes were labeled and aliquot 2.5µl of the reaction
mixture to each tube.
3. 7.5µl of the DNA sample were added to each of the digestion tubes.
4. The tubes were centrifuged, vortexed and again centrifuged to ensure
proper mixing.
5. The tubes were then incubated overnight (16hrs) in a water bath at a
recommended temperature (37°C).
6. After incubation, the digested products were loaded into the wells of the
agarose gel (3%) along with a loading dye (orange G).
7. Current was applied and after sufficient band separation, the bands were
viewed under an UV transilluminator.
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8. Then the gel picture was captured and saved in a gel doc.
30
Tables and Figures
Figure 1: Representative Gel picture showing amplification product obtained with primer after
gradient PCR
Figure 2: Representative Gel picture showing amplification product obtained with primers
31
Figure 3: Gel picture showing RFLP done for all the samples were shown to monomorphic
CC and gave two bands.
Fragment size genotype rs225089
Homozygous CC 134, 115
GG 249
Heterozygous CG 249,134,115
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Figure 4: Sequence results showing homozygous GG, heterozygous GC and homozygous CC
genotype
Figure 5: Sequence results showing homozygous GG, heterozygous GT and homozygous TT
genotype
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SECTION-B
ANIMAL CELL CULTURE
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Experiment B1
PREPARING AN ASEPTIC CONDITION
Aim
To ensure cell culture procedures are performed to a standard that will prevent
contamination from bacteria, fungi,and mycoplasma and cross contamination
with other cell lines.
Materials required
• 70% ethanol in water
Equipment
• Personal protective equipment (sterile gloves, laboratory coat)
• Microbiological safety cabinet at appropriate containment level
Procedure
1. Sanitize the cabinet using 70% ethanol before commencing work.
2. Sanitize gloves by washing them in 70% ethanol and allowing drying for 30
seconds before commencing work.
3. Put all materials and equipment into the cabinet prior to starting work after
sanitizing the exterior surfaces with 70% ethanol.
4. Discard gloves after handling contaminate cultures and at the end of all cell
culture procedures.
5. Movement within and immediately outside the cabinet must not be rapid.
Slow movement will allow the air within the cabinet to circulate properly
6. Speech,sneezing and coughing must be directed away from the cabinet so as
not to disrupt the airflow.
7. After completing work disinfect all equipment and materials before removing
from the cabinet. Spray the work surfaces inside the cabinet with 70%
ethanol and wipe with tissue paper.
8.Periodically clean the cabinet surfaces with a disinfectant
35
Method for cleaning CO2 incubator and biosafety cabinet
8.a.Clean CO2 incubator with 2.5% sodium hypochlorite
8 b. Leave for 5 min. Rinse with water and remove water completely using
tissue.
8.c.Spray incubator with 70% Isopropanol. Wipe with dry tissue to remove any
residual sodium hypochlorite and water.
36
Experiment B2
PREPARATION OF CELL GROWTH MEDIUM AND CELL GROWTH
CONDITIONS
Aim
Before starting work check the information given with the cell line to identify
what media type, additives and recommendations should be used.
Most cell lines can be grown using DMEM culture media or RPMI culture media
with 10% Fetal Bovine Serum (FBS) and antibiotics can be added if required.
Most cell lines will grow on culture flasks without the need for special matrixes
etc. However, some cells, particularly primary cells, will require growth on
special matrixes such as collagen to promote cell attachment, differentiation or
cell growth.
Materials required 1.DMEM 2.FBS 3.Antimycotic antibiotic solution Procedure a) Media preparation
i. Preparation of DMEM A. Add powdered medium to 15°C to 30 °C (Room temperature)
autoclaved water with gentle stirring.(Do not heat).
B. Rinse out inside of packet to remove traces of powder.
C. Add 1.7g of NaHCO3 per litre of medium.
D. Dilute to desired volume water. Stir until dissolved.
37
E. Adjust pH of medium to 0.2-0.3 below desired final working
ph(7-7.4). Use of NaCl or HCl is recommended. Keep container
closed until medium is filtered.
F. Sterilize immediately by membrane filtration.(Positive pressure
recommended).
b) FBS heat inactivation
1. Transfer 500ml FBS from –80 C freezer to refrigerator to thaw on. Complete
thawing of serum is done the following day by placing the serum in a 37C
water bath in which the water level is a little higher than the serum level in the
bottle. Mix by inversion after each 10 min.
2. Once the serum is completely thawed, incubate it for another 15 min to
equilibrate serum with 37 C water bath.
3. Raise the temperature setting of the bath to 56 C. Use a timer to measure
the 35 min needed for the temperature of the serum and bath to come to 56 C.
During incubation invert the bottle every 10 min to mix the serum.
4. Once the bath reaches 56 C, incubate serum for 30 min. Invert bottle ever
10 min.
5. Remove the serum from waterbath and allow to cool at room temperature for
30 min.
6. Aliquot 50ml of treated serum into conical tubes and store at 4 C or freeze at
-20 C.
c) Media preparation 1. Add the given constituents in required amounts into a 50 ml centrifuge
tube 2. Store the medium at 4 degree Celsius.
38
1.DMEM 45ml
2.10% FBS 5 ml
3.Antibiotic antimycotic solution 0.5 ml
50 ml
Providing culturing conditions
Cell lines are maintained at 370C incubator at 5% CO2.
For human cells, coat flasks with 1% gelatin.
Prepare 10mL of coating solution composed of 1% gelatin by diluting with
distilled water, followed by filtration. This is efficient to coat about 5 flasks.
1. Pipette coating solution into flask. Rock back and forth to evenly
distribute the bottom of the flask. Let sit in an incubator for 15-30
minutes.
2. Completely remove coating solution by aspirating before seeding.
39
Experiment B3
CHECKING CELLS
Cells should be checked microscopically daily to ensure they are healthy
and growing as expected. Attached cells should be mainly attached to the
bottom of the flask, round and plump or elongated in shape and refracting light
around their membrane. Suspension cells should look round and plump and
refracting light around their membrane. Some suspension cells may clump.
Media should be pinky-orange in color.
Discard cells if:
They are detaching in large numbers (attached lines) and/or look shriveled and
grainy/dark in color.
They are in quiescence (do not appear to be growing at all).
Media change is essential when the colour of the medium in culture flask turns
from red to orange (due to accumulation of toxins). Trypsinization of cells
should be done when 85-90% confluency is reached.
40
Experiment B4
SUBCULTURE OF ADHERENT CELL LINES
Aim
Adherent cell lines will grow in vitro until they have covered the surface area
available or the medium is depleted of nutrients. At this point the cell lines
should be sub-cultured in order to prevent the culture dying. Cell passaging or
splitting is a technique that enables an individual to keep cells alive and
growing under cultured conditions for extended periods of time. Cells should
be passed when they are 85%-90% confluent. To subculture the cells they need
to be brought into suspension. The degree of adhesion varies from cell line to
cell line but majority of cases proteases, e.g. trypsin ,are used to release the
cells from the flask, However, this may not be appropriate for some time where
exposure to proteases is harmful or where the enzymes are used to remove
membrane markers/receptors of interest. In these cases cells should be
brought into suspension into a small volume of medium mechanically with the
aid of cell scrappers.
Materials required
• DMEM-FBS medium
• PBS/EDTA solution
• 0.05% Trypsin
Equipment
• Personal protective equipment
• Microbiological safety cabinet
• CO2 incubator
• Pre-labeled flasks
41
• Marker pen
• Micro pipettes
• Ampule rack
Procedure
1. Discard the media from the culture flask.
2. Wash the culture flask twice with 2ml PBS/EDTA.
3. Add Trypsin and gently shake it so that cells get detached.
4. Add 1ml fresh media to deactivate trypsin
5. Transfer to a centrifuge tube
6. Centrifuge at 1000rpm for 3 min
7. Discard supernatant
8. Add 1ml fresh media and transfer to culture flask.
42
Experiment B5
COUNTING CELLS WITH HEMOCYTOMETER
a. Preparing hemocytometer
i. Ensure the hemocytometer is clean using 70% ethanol.
ii. Moisten the shoulders of the hemocytometer and affix the coverslip using
gentle pressure and small circular motions. The phenomenon of Newton’s rings
can be observed when the coverslip is correctly affixed, thus the depth of the
chamber is ensured.
b. Counting
i. Using the Gilson pipette, draw up some cell suspension containing trypan
blue. Carefully fill the haemocytometer by gently resting the end of the Gilson
tip at the edge of the chambers. Take care not to over- fill the chamber. Allow
the sample to be drawn out of the pipette by capillary action, the fluid should
run to the edges of the grooves only. Re-load the pipette and fill the second
chamber if required.
ii. Focus on the grid lines of the hemocytometer using the 10X objective of the microscope. Focus on one set of 16 corner square at the corners.
iii. Take the average of the number of cells found at the corners.
iv. Cells per ml = the average count per square x the dilution factor x 104
43
Experiment-B6 FREEZING OF CELL-LINE Aim
It is common practice to create a master bank consisting of 2 to 20 vials of the
cell line. Then create one or two working banks from this with 2 to 20 vials in
each (depending on how often the cells will be required). When the working
bank is used up, a new working bank can be cultured and created from one
vial of the original master bank. If possible, keep the master and working bank
in separate liquid nitrogen storage tanks.
This will ensure you always have a stock of cells from a lower passage number
and it will also not be necessary to keep purchasing the cell line.
Materials
1. 1 ml - 2 ml cryovial
2. Cell culture medium with 20% FBS (Fetal bovine serum) and necessary
supplements
3. DMSO (Dimethyl sulfoxide), high purity, sterile, for cell culture
4. Prepare freezing medium: to cell culture medium, add 5-10% (v/v)
DMSO.
Procedure
1. Split the cells. Take 1ml to cryovial.
2. Centrifuge at 2500rpm for 3 minutes at 4 °C.
3. Discard the supernatant and add 750ul freezing mixture (9ml FBS + 1ml
DMSO).
4. Store at -20 °C for 1 day, then in -70 °C (2-4 days) and then in liquid N2.
44
Precautions
This step must be done as soon as the cells are in freezing media. DMSO and
some other cryoprotectants are toxic to cells and so should not be exposed to
the cells at room temperature for any longer than necessary. Thawing of the
vials and placing of the cell suspension back into culture media should also be
done very quickly for the same reasons.
45
Experiment B7
REVIVAL OF CELL LINES Aim Many cultures obtained from a culture collection will arrive frozen and in order
to use them, the cells must be thawed and put into culture. It is vital to thaw
cells correctly in order to maintain the viability of the culture and enable the
culture to recover more quickly. Some cryoprotectants such as DMSO are toxic
above 4 °C. Therefore it is essential that the cultures be thawed quickly diluted
in culture medium to minimize the toxic effects.
Materials required
• Media- pre-warmed to the appropriate temperature.
Equipment required
• Personal protective equipment (sterile gloves, laboratory coat)
• Waterbath set to appropriate temperature
• Microbiological safety cabinet at appropriate containment level
• CO2 incubator
• Pre labeled flasks
• Marker pen
• Micropipettes
• Ampule rack
• Tissue
Procedure 1. Take the cells from -80 °C. Thaw it at 37 °C
2. Centrifuge at 2000 rpm for 5 min
3. Discard the supernatant.
46
4. Transfer to 15 ml tube. Add 5ml 20% DMEM
5. Centrifuge at 2000 rpm for 5 min and discard the supernatant.
6. Take 5 ml 20 % DMEM in culture flask. Transfer pellet to it
7. Start maintaining the cell line
47
Experiment B8
CELL SEEDING FOR DNA/RNA ISOLATION
Procedure
1. Discard existing media
2. Add 2 ml PBS-EDTA and wash twice
3. Add trypsin and gently swirl
4. Add 1 ml media
5. Transfer to Centrifuge tube
6. Centrifuge at 1000rpm for 3 min
7. Discard supernatant
8. Add 1ml fresh media
9. Dilute it 10 fold with media.
10. Take 10ul and count the number of cells using haemocytometer.
11. We require 3 x 105 cells in a culture plate. So, with the help of
V1N1=V2N2
We calculate the volume of media to be added to each culture plate.
12. Add [1000-(volume of cell added) ] media to each culture plate
13. Incubate at 37°C
48
Experiment B9
DRUG TREATMENT ON CULTURED CELL LINES
Aim
To subject cultured cell lines to different concentration of drug and thereby
study the effect of drug concentration on the cells.
Procedure
1. We prepare 5 different concentrations of drug- haloperidol (antipsychotic
drug; stored at -20 °C; light sensitive) by serial dilution using the formula
V1M1=V2M2
Where V1= Volume of drug to be taken
M1= Concentration of Drug given.
V2= Required Volume
M2= Required concentration
From the 1mM stock solution, 1µM, 5µM, 10µM, 15µM and 25µM drug
was prepared by serial dilution
2. Discard existing media from culture flasks
3. Add 1ml of mixture of fresh media and different concentration of drug
into culture flasks.
4. Add 1ml of DMSO containing medium (DMSO concentration < 0.1%)
into control culture flasks.
49
Experiment B10
DNA ISOLATION FROM CULTURED CELL LINE
(QIAGEN DNA isolation kit)
1. Cells suspended in DMEM stored at -20 °C
2. Remove the supernatant completely and discard
3. Add 20ul PBS to resuspend the pellet
4. Add 20ul proteinase K
5. Add 20ul buffer AL (lysis buffer) and mix by pulse vortexing for 15s
6. Incubate at 56 °C for 10 min
7. Add 200ul chilled alcohol (60-100%) & mix by pulse vortexing for 15s
8. Transfer to mini spin column & centrifuge at 8000rpm, 23-25 °C for 2
min
9. Discard the filtrate
10. Add Buffer AW1(wash buffer) 500 ul & centrifuge at 8000rpm, 23 –
25 °C for 2 minutes
11. Discard the filtrate
12. Add 500ul Buffer AW2(wash buffer)
13. Centrifuge at 14000 rpm at 25 °C for 3 min
14. Add 200ul Buffer AE(elution buffer)
15. Incubate at room temperature for 5 min
16. Centrifuge at 8000 rpm at 25 °C for 2 min
17. Store at -20 °C
50
Experiment B11
QUANTIFICATION USING NANODROP
Aim
Purity of DNA sample can also be calculated based upon its absorbance of
light. A pure sample of DNA has a 260nm/280nm ratio of 1.8. Ratios deviating
from this usually indicate contamination of the sample with proteins, organic
solvents or RNA or could indicate degradation of the DNA sample.
Materials Required
1.Nanodrop
Procedure
1. Clean the sample loading point with sterile water to initialize the
equipment
2. Open the bundled software
3. Add 1ul of nuclease free water solution in which DNA is dissolved and
calculate Blank’
4. Now clean the loading point and load subsequent samples and measure
quantification.
5. Record the values of DNA concentration(ng/ul) and A260/A280
51
Experiment B12
RNA ISOLATION FROM CULTURED CELL LINES (TRIZOL
METHOD)
Procedure
1. Wash cells with 1X PBS.
2. Add 1ml TRIzol per well.
3. Incubate at room temperature for 3 minutes
4. Add 200ul CHCl3
5. Pulse vortex for 15 seconds and incubate at room temperature for 3
minutes.
6. Centrifuge for 15 minutes at 8000 rpm at 4 °C.
7. Pipette water phase into a new eppendorf tube.
8. Add 500ul isopropanol per ml of trizol
9. Incubate at -20 °C for 30minutes.
10.Centrifuge for 10 minutes at 14000rpm at 4 °C
11.Wash pellet with 500ul 70% ethanol.
12. Centrifuge for 5 min at 14000rpm at room temperature.
13. Air dry the pellet (20 min)
14. Resuspend in 30-50ul nuclease-free water.
15. Keep at 56 °C for 10 minutes
16.Store at -80°C.
52
Experiment- B13
ASSESSMENT OF QUALITY AND QUANTITY OF RNA.
a) Quality of RNA can be checked by agarose electrophoresis followed by
ethidium bromide staining. 2µl of RNA is run on 1.2% agarose gel and
photographed.
The presence of crisp bands corresponding to 28 S rRNA and 18 S rRNA
indicates the quality of the RNA isolated.
b) RNA concentration can be measured using NanoDropTM
spectrophotometer.
A260/A280 ratio for RNA should be 1.8-2. A260/A230 ratio should be 2-2.2
Procedure
1. Clean the sample loading point with sterile water.
2. Open the bundled software
3. Add 1ul of nuclease free water solution in which RNA is dissolved and
calculate Blank’
4. Now clean the loading point and load subsequent samples and measure
quantification.
5. Record the values of RNA concentration A260/A280 and A260/A230.
53
Experiment B14
cDNA PREPARATION
Procedure
1. Prepare a mixture in an eppendorf tube with the following constituents
10 x RT buffer 2 ul
25X dNTP 0.8 ul
10 X random primer 2 ul
Reverse transcriptase 1 ul
Nuclease Free Water 4.2 ul
10ul
2. Prepare 1 ug of RNA in 10 ul
3. Mix 10ul of RNA and 10 ul of reaction mix.
4. Place the tube in a thermal cycler and run the following program
25 oC 10 min
37 oC 120 min
85 oC 5 min
4 oC infinity
QUALITY CONTROL TEST FOR cDNA
This test was being performed by PCR amplification of β-Actin.
Reaction mixture was prepared with following components
cDNA - 1µL
54
10XRT buffer - 1µL P.T.O
2.5mMdNTP - 1µL
20 uM forward primer - 0.09ul
20 uM reverse primer - 0.09ul
Taq polymerase - 0.2ul
H2O - 6.62ul
10ul
PCR conditions
95°`C 3 min
95°C 30 sec
56.7°C 15 sec
72°C 30 sec
72°C 10 min
4°C ∞
Following PCR,the product was run in 1% gel and analysed for bands
corresponding to B-actin.
55
Experiment B15
REAL-TIME PCR
Aim
This technique is used to amplify and simultaneously quantify a
targeted DNA molecule. Here the amplified DNA is detected as the reaction
progresses in real time. Two common methods for detection of products in real-
time PCR are: (1) non-specific fluorescent dyes that intercalate with any
double-stranded DNA, and (2) sequence-specific DNA probes consisting
of oligonucleotides that are labeled with a fluorescent reporter which permits
detection only after hybridization of the probe with its complementary DNA
target.
56
There are basically two methods of analyzing the data from a real time PCR-
absolute quantification and relative quantification. Relative quantification or
comparative quantification measures the relative change in mRNA expression
levels. It determines the changes in steady state mRNA levels of a gene across
multiple samples and expresses it relative to the levels of another RNA. Relative
concentrations of DNA present during the exponential phase of the reaction are
determined by plotting fluorescence against cycle number on a logarithmic
scale (so that an exponentially-increasing quantity will show as a straight line).
A threshold for detection of fluorescence above background is determined. The
cycle at which the fluorescence from a sample crosses the threshold is called
the cycle threshold, Ct.
The amount of target, normalized to an endogenous reference and relative to a
calibrator is given by,
Amount of target = 2-ΔΔCT
Where ΔΔCT = (CT,target – CT,actin )time,x – (CT,target – CT,actin)time,0.
Here time ‘x’ is any time point and time’0’ represents the expression of the
target gene normalized to β-actin.
Procedure
PCR components for TaqMan based gene expression assay
cDNA - 2µl
TaqMan Universal PCR Master Mix - 5 µl
20X TaqMan Gene Expression Assay Mix - 0.5 µl
Nuclease-free water - 2.5 µl
Total - 10 µl
57
Reaction mix was prepared for B-actin also, which served as the reference
gene.
The samples were loaded on 96-well plate and each sample was run in
triplicates.
The plate was run on the ABI 7900HT with the following settings:
Step 1. 2 minutes at 50 C; Step 2. 10 minutes at 95 C and then 40 cycles of
Step 3.15 sec at 95 C and then 1 minute at 60 C.
The results were analyzed using SDS RQ Manager software.
.
58
Reference:
Section A Molecular markers, Natural history and Evolution -‐ John.C.Avis
Calculations for Molecular Biology and Biotechnology – Frank.H.Stephen
Principles of Gene manipulation – Sandy B. Primrose, Richard M. Twyman, Robert W. Old
http://humgen.wustl.edu/hdk_lab_manual/pcr/pcr1.html
http://www.dnalc.org/resources/animations/gelelectrophoresis.html
http://www.biocompare.com/Articles/ApplicationNote/648/Using-‐Gradient-‐PCR-‐To-‐
Determine-‐The-‐Optimum-‐Annealing-‐Temperature.html
Section B http://www.research.umbc.edu/~jwolf/method1.htm
Sigma-Aldrich ECACC handbook
http://www.tissue-‐cell-‐culture.com/docs/libary/tc_trouble_shooting.pdf
http://www.ruf.rice.edu/~bioewhit/labs/bioe342/docs/cell%20passage.htm www.abcam.com/index.html?pageconfig=resource&rid=11455 www.abcam.com/index.html?pageconfig=resource&rid=11742 www.protocol-‐online.org/biology-‐forums/cell-‐culture.html www.abcam.com/index.html?pageconfig=resource&rid=11453 www.userpages.umbc.edu/~jwolf/method5.htm