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Rajiv Gandhi Centre for Biotechnology Department of Biotechnology, M inistry of Science & Technology, Thiruvananthapuram, Kerala

SNP Genotyping and Cell Culture Techniques Human Molecular Genetics Lab

Submitted by Stevin Wilson Aruna Mohan

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.

Contents
Section-A: SNP Genotyping
A1.DNA ISOLATION 6 A2.DNA QUANTIFICATION AND QUALITY CHECK OF DNA A3.POLYMERASE CHAIN REACTION 13 10

A4.AGAROSE GEL ELECTROPHORESIS 19 A5.DNA SEQUENCING A6.RESTRICTION FRAGMENT LENGTH POLYMORPHISM TABLES AND FIGURES 30 27 21

Section-B: Cell Culturing


B1.PREPARING AN ASEPTIC ENVIRONMENT 34 B2.PREPARATION OF CELL GROWTH MEDIUM AND CULTURE CONDITIONS 36 3

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

Section-A SNP GENOTYPING- BASIC TECHNIQUES

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 56c 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 6

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: Buffers used: RBC lysis buffer (Tris: EDTA: NaCl-30: 5:50mM) WBC lysis buffer (NaCl: EDTA-75: 2mM) T.E buffer (Tris: EDTA10: 1mM) 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

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) 7

0.5M EDTA 1 ml 5 M NaCl TE buffer: (10:1mM) 1M Tris 1 ml 0.5M EDTA - 0.2 ml Distilled water 100 ml Equipment and instruments: o Deep freezer (-80C) 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 Day2 Remove the blood samples from freezer and thaw it in a water bath for 10-15 minutes. Intravenous blood specimen collected in EDTA tubes can be stored for a longer period of time at -20c or -80c for later extraction of DNA. In such specimens equal volume of RBC Lysis buffer is added. - 3.75 ml Distilled water - 250 ml

Centrifuge the tubes at 10,000rpm for 10minutes at 15c. 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 100 /ml and SDS to make 2% concentration in the final volume. Mix well and incubate the samples at 37 c 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 4c. 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. 9 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 4c or -20c or-80c for future use.

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-Lamberts law that gives the linear relationship between absorbance and concentration of absorbing species: A= b c Where A is measured absorbance, 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. 10

Based on its structure, DNA absorbs light in the ultraviolet range, specifically at a wavelength of 260nm. A value of 1 at OD is equal to 50ng/l doublestranded DNA, therefore to calculate the concentration of DNA ; the following formula can be used: Concentration DNA =260nmabs 50ng/ l 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 l of DNA preparation and dilute it to 100 l with Double Distilled water and mix well. The spectrophotometer is calibrated at 260nm and 280nm with 100 l 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 50g/ml. So DNA concentration of the extracted sample = OD at 260nm 50 Dilution factor 12

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 13

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. 14

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 5C 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 (>12C). 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 64C, 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 DNA 10X Buffer dNTPs 20 pM forward primer 20 pM reverse primer Taq polymerase (3U) Sterile water Total

VOLUME(l) 1 1 1 0.1 0.1 0.12 6.68 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 72C 72C 4o C 94C 94C 550C to 65oC 35Cycles 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 Distilled water 10x Buffer 2.5M dntp 20pM Forward Primer Quantity per PCR tube in l 6.68 1.0 1.0 0.1 16

20pM Reverse Primer 5U/l Taq Polymerase(NEB)

0.1 0.12

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 l 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 l 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:

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Initial denaturation Denaturation Annealing Extension Final extension Hold

94C 94C 550C 72C 72C 4o C

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 3045minutes, 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 l of Ethidium bromide into the mixture and the mix was poured into the gel tray taped on all sides. 19

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, 3dideoxynucleotides, 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, 3dideoxynucleotide 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. 22

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 23

The following cocktail reaction mixture was prepared for the required number of reactions.

Components DNA (PCR product) Sequencing Buffer (5X) Sequence mix Primer (forward) Sterile water Total Sequencing PCR reaction mixture i. ii. iii. iv. Prepare sufficient sequencing master mix. Vortex and centrifuge the master mix briefly.

Volume 1 l 2l 0.25l 0.4l 6.35 10l

Add 9.5l of master mix to 0.5l of cleaned PCR product. Place the samples on the PCR thermocycler using the following conditions:

The Sequencing PCR is carried out as in condition shown below PCR steps Denaturation Extension Hold TEMPERATURE 94C(35 cycles) 60C 4C TIME 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,

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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 10l water and 2 l of 125mM EDTA to each sample and mix. 2. Add 2l 3M sodium acetate (pH 5.2) and 50 l 100% ethanol to each sample. Incubate for 15minutes at room temperature. 3. Centrifuge for 12,000rpm for 20minutes at 26c. 4. Decant the supernatant, add 100 l 70% ethanol and centrifuge at 12,000rpm for 10 minutes at 26c.Repeat the step for once more. 5. Decant the supernatant and air-dry the pellet at room temperature. 6. Add 10l formamide to each DNA pellet and seal the plate. 7. Denature samples by heating to 96c 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 27

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 Sterile distilled water Buffer (NEB 2) Enzyme Quantity 1.25l 1.0l 0.25l

1. This mixer was first vortexed and spinned down using a micro centrifuge 2. The digestion tubes were labeled and aliquot 2.5l of the reaction mixture to each tube. 3. 7.5l 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 (37C). 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. 28

8. Then the gel picture was captured and saved in a gel doc.

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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

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Fragment size Homozygous Heterozygous

genotype rs225089 CC GG CG 134, 115 249 249,134,115

Figure 3: Gel picture showing RFLP done for all the samples were shown to monomorphic CC and gave two bands.

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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 Personal protective equipment (sterile gloves, laboratory coat) Microbiological safety cabinet at appropriate containment level Equipment

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

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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.

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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 15C 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.

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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.

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1.DMEM 2.10% FBS 3.Antibiotic antimycotic solution

45ml 5 ml 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.

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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.

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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 Personal protective equipment Microbiological safety cabinet CO2 incubator Pre-labeled flasks 40

Equipment

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.

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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 Newtons 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

42

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. 43

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.

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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. 45

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

46

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 37C

47

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, 1M, 5M, 10M, 15M and 25M 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.

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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. 11. 12. 13. 14. 15. 16. 17. Add Buffer AW1(wash buffer) 500 ul & centrifuge at 8000rpm, 23 Discard the filtrate Add 500ul Buffer AW2(wash buffer) Centrifuge at 14000 rpm at 25 C for 3 min Add 200ul Buffer AE(elution buffer) Incubate at room temperature for 5 min Centrifuge at 8000 rpm at 25 C for 2 min Store at -20 C 25 C for 2 minutes

49

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

50

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 -80C.

51

Experiment- B13 ASSESSMENT OF QUALITY AND QUANTITY OF RNA.


a) Quality of RNA can be checked by agarose electrophoresis followed by ethidium bromide staining. 2l 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.

52

Experiment B14 cDNA PREPARATION


Procedure 1. Prepare a mixture in an eppendorf tube with the following constituents 10 x RT buffer 25X dNTP 10 X random primer Reverse transcriptase Nuclease Free Water 2 ul 0.8 ul 2 ul 1 ul 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 -Actin. Reaction mixture was prepared with following components
cDNA - 1L

53

10XRT buffer 2.5mMdNTP 20 uM forward primer 20 uM reverse primer Taq polymerase H 2O

- 1L - 1L - 0.09ul - 0.09ul - 0.2ul - 6.62ul 10ul

P.T.O

PCR conditions 95`C 3 min 95C 30 sec

56.7C 15 sec 72C 30 sec

72C 10 min 4C

Following PCR,the product was run in 1% gel and analysed for bands corresponding to B-actin.

54

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 realtime 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.

55

There are basically two methods of analyzing the data from a real time PCRabsolute 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-CT Where CT = (CT,target CT,actin )time,x (CT,target CT,actin)time,0. Here time x is any time point and time0 represents the expression of the target gene normalized to -actin. Procedure PCR components for TaqMan based gene expression assay cDNA TaqMan Universal PCR Master Mix 20X TaqMan Gene Expression Assay Mix Nuclease-free water Total 2l 5 l

- 0.5 l - 2.5 l 10 l

56

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. .

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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 58