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  • 1. Rajiv Gandhi Centre for Biotechnology Department of Biotechnology, M inistry of Science & Technology, Thiruvananthapuram, KeralaSNP Genotyping and Cell Culture TechniquesHuman Molecular Genetics Lab Submitted by Stevin Wilson Aruna Mohan    
  • 2. AcknowledgementWe would like to express our gratitude to all those who gave us the possibility tocomplete this project.First of all we would like to thank Dr.Radhakrishnan Pillai, Director, Rajiv Gandhi Centrefor 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 outstandingsupport and knowledge he shared with us.This report would not have been possible without the help of Ms. Swathy B, Mr.SanishSathyan, Ms. Sarada Lakshmi,Mr. Shabeesh Balan, Mr. Antony KP and Ms. AnaswaraAshok. They steered us through the basics of all techniques practiced in the lab. Wewould 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 thetenure.Above all we thank the Almighty for His blessings without which this training would havebeen just a dream. 2    
  • 3. ContentsSection-A: SNP GenotypingA1.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 CulturingB1.PREPARING  AN  ASEPTIC  ENVIRONMENT                                                                                                                                                    34    B2.PREPARATION  OF  CELL  GROWTH  MEDIUM  AND  CULTURE  CONDITIONS                                                                                                  36     3    
  • 4. 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   4    
  • 5.     Section-A SNP GENOTYPING- BASIC TECHNIQUES 5    
  • 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 ofsufficient amounts of DNA of the appropriate quality. Many different methodsand technologies are available for the isolation of genomic DNA. All methodsinvolve disruption and lysis of the starting material followed by the removal ofproteins and other contaminants and finally recovery of the DNA.The method used here is “Phenol –chloroform DNA extraction’’ from bloodsample. This method was found by was originally devised by Piotr Chomczynskiand Nicoletta Sacchi and published in 1987 (referred to as Guanidiniumthiocyanate-phenol-chloroform extraction).Principle: Human blood consists of enucleated RBCs and nucleated WBCs andplatelets. The blood is first suspended in RBC lysis buffer. This buffer consistsof 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 nucleasesthus inhibiting their activity. The pellet obtained after centrifugation consistsmainly 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 nuclearmembranes are also broken down by the action of the blender. Incubation at56⁰c in the presence of Proteinase K and SDS is used to partially digestcellular proteins and loosen the association between proteins and chromosomalDNA and to degrade cellular RNA.The cell lysate is then treated with buffersaturated phenol: chloroform. The DNA remains in the aqueous phase whilethe cellular proteins are extracted into the organic phase. The aqueous phase 6    
  • 7. is often extracted a second time with phenol: chloroform to ensure completeremoval of the proteins. The aqueous phase, containing the DNA, is thenwashed with ethanol and then dissolved in water or TE Buffer. DNA extractedthis way is generally high molecular weight and double stranded is thereforesuitable 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) 7    
  • 8. − 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 mlEquipment 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 PipettesProcedureDay 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. 8    
  • 9. • 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.       9    
  • 10. Experiment-A2 DNA QUANTITATION AND QUALITY CHECK OF DNA Prior to any analysis, DNA samples should be quantitated and checkedfor purity of DNA. The amount of light that a sample absorbs at a particularwavelength is measured and used to determine the concentration of the sample bycomparison with appropriate standards or reference data. The most commonlyused methodologies for quantifying the amount of nucleic acid in a preparationare: (i) gel electrophoresis and (ii) spectrophotometric analysis. Here the sampleamount being less, the latter method is preferred.a) Spectrophotometric DeterminationThe spectrophotometric quantification of DNA is based on Beer-Lambert’s law thatgives the linear relationship between absorbance and concentration of absorbingspecies: A= λ × b × cWhere A is measured absorbance, λ is wavelength dependent absorptivitycoefficient, b is path length and c is analyte concentration.Analysis of UV absorption by the nucleotides provides a simple and accurateestimation of the concentration of nucleic acids in a sample. Purines andpyramidines in nucleic acid show absorption maxima around 260nm (e.g. dATP :259nm ; dCTP : 272nm ; dTTP : 247nm) if the DNA sample is pure withoutsignificant contamination from proteins or organic solvents. The ratio ofOD260/OD280 genomic DNA should be determined to assess the purity of thesample. This method is however limited by the quantity of DNA and the purity ofthe preparation. Accurate analysis of the DNA preparation may be impeded by thepresence of impurities in the sample or if the amount of DNA is too little. In theestimation of total genomic DNA, for example, the presence of RNA, sheared DNAetc. could interfere with the accurate estimation of total high molecular weight. 10    
  • 11. Based on its structure, DNA absorbs light in the ultraviolet range, specificallyat 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 followingformula can be used: Concentration DNA =260nmabs × 50ng/ µlPurity of DNA sample can also be calculated based upon its absorbance oflight. A pure sample of DNA has a 260nm/280nm ratio of 1.8. Ratios deviatingfrom this usually indicate contamination of the sample with proteins, organicsolvents or RNA or could indicate degradation of the DNA sample.ProcedureDetermination 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 extractedDNA sample. If the ratio is within 1.6 to 2 range, the DNA is considered asclear and free from contaminants.An OD ratio less than 1.6 indicate the residual proteins or phenolcontamination, whereas ratio of more than 2.0 indicates residual RNAcontamination. 11    
  • 12. Quantity Assessment:If the OD value at 260nm of extracted sample is 1.00, then the concentration ofDNA is 50µg/ml.So DNA concentration of the extracted sample = OD at 260nm × 50 × Dilutionfactor                                                                       12    
  • 13.         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 otherapplications. The name, polymerase chain reaction, comes from the DNApolymerase 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 thesemolecules is replicated in a second "cycle" of replication, resulting in four timesthe number of the original molecules. Again, each of these molecules isreplicated in a third cycle of replication. This process is known as a "chainreaction" in which the original DNA template is exponentially amplified. WithPCR it is possible to amplify a single piece of DNA, or a very small number ofpieces of DNA, over many cycles, generating millions of copies of the originalDNA molecule. PCR has been extensively modified to perform a wide array ofgenetic manipulations, diagnostic tests, and for many other uses.Applications:The polymerase chain reaction is used by a wide spectrum of scientists in anever-increasing range of scientific disciplines. In microbiology and molecularbiology, for example, PCR is used in research laboratories in DNA cloningprocedures, Southern blotting, DNA sequencing, recombinant DNA technology,to name but a few. In clinical microbiology laboratories PCR is invaluable forthe diagnosis of microbial infections and epidemiological studies. PCR is alsoused in forensics laboratories and is especially useful because only a tiny 13    
  • 14. amount of original DNA is required, for example, sufficient DNA can beobtained from a droplet of blood or a single hair.PrincipleThe polymerase chain reaction (PCR) is a method for oligonucleotide primerdirected enzymatic amplification of a specific DNA sequence of interest. Thistechnique is capable of amplifying a sequence 105 to 106-fold from nanogramamounts of template DNA within a large background of irrelevant sequences(e.g. from total genomic DNA). A prerequisite for amplifying a sequence usingPCR is to have known, unique sequences flanking the segment of DNA to beamplified so that specific oligonucleotides can be obtained. It is not necessaryto know anything about the intervening sequence between the primers. ThePCR product is amplified from the DNA template using a heat-stable DNApolymerase from Thermus aquaticus (Taq DNA polymerase) and using anautomated thermal cycler to put the reaction through 30 or more cycles ofdenaturing, annealing of primers, and polymerization.After amplification by PCR, the products are separated by polyacrylamide gelelectrophoresis and are directly visualized after staining with ethidiumbromide. Ethidium bromide is added to the agarose to stain the DNA. Ethidiumbromide, a fluorescent dye binds tightly to the DNA double helix and glowswhen illuminated with ultraviolet light. This lets us see where the separatedDNA fragments end up.Primer Design Online toolsNCBI database - reference, database of SNP, Genes etc.UCSC – Insilco PCRPrimer 3 – to design primerGene 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    
  • 15. 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 PCRThe selection of the annealing temperature is possibly the most criticalcomponent for optimizing the specificity of a PCR reaction. In most cases, thistemperature must be empirically tested. The PCR is normally started at 5°Cbelow the calculated temperature of the primer melting point (Tm). However,the possible formation of unspecific secondary bands shows that the optimumtemperature is often much higher than the calculated temperature (>12°C).Further PCR reactions with gradually increasing temperatures are requireduntil the most stringent conditions have been found. When a standard PCRcycler is used, this method is the most time-intensive optimization strategy. During the PCR, a temperature gradient, which can be programmed betweensay 50 and 64°C, is built up across the thermo block. This allows the moststringent parameters for every primer set to be calculated with the aid of onlyone single PCR reaction.The following reaction mixture was prepared for a required number ofreactions: 15    
  • 16. COMPONENTS VOLUME(µl)DNA 110X Buffer 1dNTPs 120 pM forward primer 0.120 pM reverse primer 0.1Taq polymerase (3U) 0.12Sterile water 6.68Total 10Vortex the mixture briefly, then centrifuge at low speed. The Gradient PCR wasperformed with the above tubes in a thermal cycle according to the followingprotocolInitial denaturation 94°C 5 minutesDenaturation 94°C 30seconds 35Cycles    Annealing 550C to 65oC 30secondsExtension 72°C 30secondsFinal extension 72°C 5 minutesHold 4o C ForeverProtocol 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 16    
  • 17. 20pM Reverse Primer 0.1 5U/µl Taq Polymerase(NEB) 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 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: 17    
  • 18. Initial denaturation 94°C 5 minutesDenaturation 94°C 30secondsAnnealing 550C 30secondsExtension 72°C 30secondsFinal extension 72°C 5 minutesHold 4o C 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. 18    
  • 19. Experiment-A4 GEL ELECTROPHORESISIt is used to separate DNA fragments. Electrophoresis uses an electric currentto separate different-sized molecules in a porous, sponge-like matrix. Smallermolecules move more easily through the gel pores than larger molecules.The technique uses an agarose gel, made from highly purified seaweed. Thiscould be used to separate DNA molecules ranging from several hundrednucleotides in length to over 10,000 nucleotides.Materials required ∗ Horizontal gel electrophoresis ∗ Gel tray ∗ Gel combs ∗ Power supply unit ∗ Micro wave oven ∗ UV transilluminatorReagents 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. 19    
  • 20. 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. 20    
  • 21. Experiment-A5 DNA SEQUENCINGPrincipleThe principles of DNA replication were used by Sanger et al. (1974) in thedevelopment of the process now known as Sanger dideoxy sequencing. Thisprocess takes advantage of the ability of DNA polymerase to incorporate 2′, 3′-dideoxynucleotides, nucleotide base analogs that lack the 3,′-hydroxyl groupessential in phosphodiester bond formation. Sanger dideoxy sequencingrequires a DNA template, a sequencing primer, DNA polymerase, nucleotides(dNTPs), dideoxynucleotides (ddNTPs), and reaction buffer.Four separate reactions are set up, each containing radioactively labelednucleotides and either ddA, ddC, ddG, or ddT. The annealing, labeling, andtermination steps are performed on separate heat blocks. DNA synthesis isperformed at 37 °C, the temperature at which the T7 DNA polymerase used hasthe optimal enzyme activity.DNA polymerase adds either a deoxynucleotide or the corresponding 2′, 3′-dideoxynucleotide at each step of chain extension. Whether a deoxynucleotideor a dideoxynucleotide is added depends on the relative concentration of bothmolecules. When a deoxynucleotide (A, C, G, or T) is added to the 3′ end, chainextension can continue. However, when a dideoxynucleotide (ddA, ddC, ddG, orddT) is added tothe 3´ end, chain extension terminates . Sanger dideoxy sequencing results inthe formation of extension products of various lengths terminated withdideoxynucleotides at the 3′ end. 21    
  • 22. DNA Template Preparation:*PCR Strategies: Because cycle sequencing involves many cycles of templatedenaturation and extension, adequate signal is produced in the sequencingreaction. In selecting the strategy for generating PCR DNA templates to be usedfor cycle sequencing, considering specificity and yield.*Primer design and quantitation:When you perform dye terminator cycle sequencing reactions on PCR template,theprimer sequence, primer synthesis method, and primer purification methodcangreatly affect the quality of the sequencing data.*Optimizing Primer Design:• Primers should be at least 18 bases long to ensure good hybridization and tominimize 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 lowerTm.• 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%. Forprimers with G-C content less than 50%, you may need to increase the primerlength beyond 18 bases to maintain a Tm>45 °C. 22    
  • 23. • 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 sitesand reagents in the sequencing reaction. Additional primers in sequencingreactions using dye terminators result in the creation of multiple dye-labeledsequence 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 PCRproducts. Nonspecific PCR products behave as templates in the sequencingreaction and cause the generation of multiple dye-labeled sequence ladders,which result in noisy data. Any significant quantity of nonspecific PCR productscan 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 PCRFor sequencing PCR, following constituent are needed, DNA(amplified productfrom the primary PCR),Primer (Forward or reverse),Sequence mix ,Sequencebuffer and Water 23    
  • 24. The following cocktail reaction mixture was prepared for the required number of reactions.Components VolumeDNA (PCR product) 1 µlSequencing Buffer (5X) 2µlSequence mix 0.25µlPrimer (forward) 0.4µlSterile water 6.35Total 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 belowPCR steps TEMPERATURE TIMEDenaturation 94C(35 cycles) 10 secExtension 60°C 4 minHold 4°C Foreverb) Sequencing Reaction Clean Up:Sequencing clean up is mainly done to purify the single stranded or doublestranded DNA product from primers, nucleotides, polymerases, oil and salt, 24    
  • 25. dNTPs, enzymes, short, failed PCR product so that they do not interfere withthe 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 • FormamideProcedure: 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 25    
  • 26. AB1 prism 3730 Genetic analyzer is an multi capillary automated system tosequence, size and quantitate nucleic acids using multicolour fluorescentlabeling technology .AB1 PRISM Genetic analyzer software provides thesequence data in the form of a chromatogram, where each nucleotide isrepresented as a peak; C is represented by a blue peak, A by green, G by blackand T by red.                     26    
  • 27. Experiment -A6 Restriction Fragment Length PolymorphismRestriction Fragment Length Polymorphism (RFLP) is a difference inhomologous DNA sequences that can be detected by the presence of fragmentsof different lengths after digestion of the DNA samples in question withspecific restriction endonucleases. RFLP, as a molecular marker, is specific to asingle clone/restriction enzyme combination. RFLP is one technique used byforensic scientists inDNA fingerprinting. It is also used for tracing ancestry,studying evolution and migration of wildlife, and detection and diagnosis ofcertain diseases. Most RFLP markers are co-dominant (both alleles inheterozygous sample will be detected) and highly locus-specific.The RFLP probes are frequently used in genome mapping and in variationanalysis (genotyping, forensics, paternity tests, hereditary disease diagnostics,etc.).RFLP methodology involves cutting a particular region of DNA with knownvariability, with restriction enzymes, then separating the DNA fragmentsby agarose gel electrophoresis and determining the number of fragments andrelative sizes. The pattern of fragment sizes will differ for each individual tested.Materials required  DNA sample  Enzyme buffer  Restriction enzyme  Sterile distilled waterInstrumentation  Water bath  Agarose gel electrophoresis 27    
  • 28.  Digestion tubes  Micro centrifuge  Vortex mixerProcedure 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 ofreactions. 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. 28    
  • 29. 8. Then the gel picture was captured and saved in a gel doc. 29    
  • 30. Tables and FiguresFigure  1:  Representative  Gel  picture  showing  amplification  product  obtained  with  primer  after  gradient  PCR      Figure    2:  Representative  Gel  picture  showing  amplification  product  obtained  with  primers     30    
  • 31.   Fragment  size   genotype   rs225089     CC   134,  115     Homozygous   GG   249     Heterozygous   CG   249,134,115      Figure  3:  Gel  picture  showing  RFLP  done  for  all  the  samples  were  shown  to  monomorphic  CC  and  gave  two  bands.           31    
  • 32. 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     32    
  • 33. SECTION-B ANIMAL CELL CULTURE 33    
  • 34. Experiment B1 PREPARING AN ASEPTIC CONDITIONAimTo ensure cell culture procedures are performed to a standard that will preventcontamination from bacteria, fungi,and mycoplasma and cross contaminationwith other cell lines.Materials required • 70% ethanol in waterEquipment • Personal protective equipment (sterile gloves, laboratory coat) • Microbiological safety cabinet at appropriate containment levelProcedure1. 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 properly6. 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 34    
  • 35. Method for cleaning CO2 incubator and biosafety cabinet8.a.Clean CO2 incubator with 2.5% sodium hypochlorite8 b. Leave for 5 min. Rinse with water and remove water completely usingtissue.8.c.Spray incubator with 70% Isopropanol. Wipe with dry tissue to remove anyresidual sodium hypochlorite and water.       35    
  • 36. Experiment B2 PREPARATION OF CELL GROWTH MEDIUM AND CELL GROWTH CONDITIONSAimBefore starting work check the information given with the cell line to identifywhat media type, additives and recommendations should be used.Most cell lines can be grown using DMEM culture media or RPMI culture mediawith 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 matrixesetc. However, some cells, particularly primary cells, will require growth onspecial matrixes such as collagen to promote cell attachment, differentiation orcell growth.Materials required1.DMEM2.FBS3.Antimycotic antibiotic solutionProcedure 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. 36    
  • 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 inactivation1. Transfer 500ml FBS from –80 C freezer to refrigerator to thaw on. Completethawing of serum is done the following day by placing the serum in a 37Cwater bath in which the water level is a little higher than the serum level in thebottle. Mix by inversion after each 10 min.2. Once the serum is completely thawed, incubate it for another 15 min toequilibrate serum with 37 C water bath.3. Raise the temperature setting of the bath to 56 C. Use a timer to measurethe 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 ever10 min.5. Remove the serum from waterbath and allow to cool at room temperature for30 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. 37    
  • 38. 1.DMEM 45ml2.10% FBS 5 ml3.Antibiotic antimycotic solution 0.5 ml 50 mlProviding culturing conditionsCell 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 withdistilled 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.   38    
  • 39. Experiment B3 CHECKING CELLS Cells should be checked microscopically daily to ensure they are healthyand growing as expected. Attached cells should be mainly attached to thebottom of the flask, round and plump or elongated in shape and refracting lightaround their membrane. Suspension cells should look round and plump andrefracting 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 andgrainy/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 turnsfrom red to orange (due to accumulation of toxins). Trypsinization of cellsshould be done when 85-90% confluency is reached. 39    
  • 40. Experiment B4 SUBCULTURE OF ADHERENT CELL LINESAimAdherent cell lines will grow in vitro until they have covered the surface areaavailable or the medium is depleted of nutrients. At this point the cell linesshould be sub-cultured in order to prevent the culture dying. Cell passaging orsplitting is a technique that enables an individual to keep cells alive andgrowing under cultured conditions for extended periods of time. Cells shouldbe passed when they are 85%-90% confluent. To subculture the cells they needto be brought into suspension. The degree of adhesion varies from cell line tocell line but majority of cases proteases, e.g. trypsin ,are used to release thecells from the flask, However, this may not be appropriate for some time whereexposure to proteases is harmful or where the enzymes are used to removemembrane markers/receptors of interest. In these cases cells should bebrought into suspension into a small volume of medium mechanically with theaid of cell scrappers.Materials required • DMEM-FBS medium • PBS/EDTA solution • 0.05% TrypsinEquipment • Personal protective equipment • Microbiological safety cabinet • CO2 incubator • Pre-labeled flasks 40    
  • 41. • Marker pen • Micro pipettes • Ampule rackProcedure 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.                               41    
  • 42. Experiment B5 COUNTING CELLS WITH HEMOCYTOMETERa. Preparing hemocytometeri. Ensure the hemocytometer is clean using 70% ethanol.ii. Moisten the shoulders of the hemocytometer and affix the coverslip usinggentle pressure and small circular motions. The phenomenon of Newton’s ringscan be observed when the coverslip is correctly affixed, thus the depth of thechamber is ensured.b. Countingi. Using the Gilson pipette, draw up some cell suspension containing trypanblue. Carefully fill the haemocytometer by gently resting the end of the Gilsontip at the edge of the chambers. Take care not to over- fill the chamber. Allowthe sample to be drawn out of the pipette by capillary action, the fluid shouldrun to the edges of the grooves only. Re-load the pipette and fill the secondchamber if required.ii. Focus on the grid lines of the hemocytometer using the 10X objective of themicroscope. 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    
  • 43.                                                                                           Experiment-B6 FREEZING OF CELL-LINEAimIt is common practice to create a master bank consisting of 2 to 20 vials of thecell line. Then create one or two working banks from this with 2 to 20 vials ineach (depending on how often the cells will be required). When the workingbank is used up, a new working bank can be cultured and created from onevial of the original master bank. If possible, keep the master and working bankin separate liquid nitrogen storage tanks.This will ensure you always have a stock of cells from a lower passage numberand 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.Procedure1. 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 + 1mlDMSO).4. Store at -20 °C for 1 day, then in -70 °C (2-4 days) and then in liquid N2. 43    
  • 44. PrecautionsThis step must be done as soon as the cells are in freezing media. DMSO andsome other cryoprotectants are toxic to cells and so should not be exposed tothe cells at room temperature for any longer than necessary. Thawing of thevials and placing of the cell suspension back into culture media should also bedone very quickly for the same reasons. 44    
  • 45. Experiment B7 REVIVAL OF CELL LINESAimMany cultures obtained from a culture collection will arrive frozen and in orderto use them, the cells must be thawed and put into culture. It is vital to thawcells correctly in order to maintain the viability of the culture and enable theculture to recover more quickly. Some cryoprotectants such as DMSO are toxicabove 4 °C. Therefore it is essential that the cultures be thawed quickly dilutedin 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 • TissueProcedure1. Take the cells from -80 °C. Thaw it at 37 °C2. Centrifuge at 2000 rpm for 5 min3. Discard the supernatant. 45    
  • 46. 4. Transfer to 15 ml tube. Add 5ml 20% DMEM5. Centrifuge at 2000 rpm for 5 min and discard the supernatant.6. Take 5 ml 20 % DMEM in culture flask. Transfer pellet to it7. Start maintaining the cell line 46    
  • 47. Experiment B8 CELL SEEDING FOR DNA/RNA ISOLATIONProcedure 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 47    
  • 48. Experiment B9 DRUG TREATMENT ON CULTURED CELL LINESAimTo subject cultured cell lines to different concentration of drug and therebystudy 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. 48    
  • 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 49    
  • 50. Experiment B11 QUANTIFICATION USING NANODROPAimPurity of DNA sample can also be calculated based upon its absorbance oflight. A pure sample of DNA has a 260nm/280nm ratio of 1.8. Ratios deviatingfrom this usually indicate contamination of the sample with proteins, organicsolvents or RNA or could indicate degradation of the DNA sample.Materials Required1.NanodropProcedure 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    
  • 51. Experiment B12 RNA ISOLATION FROM CULTURED CELL LINES (TRIZOLMETHOD)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. 51    
  • 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 2-2.2Procedure 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    
  • 53. Experiment B14 cDNA PREPARATIONProcedure 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  infinityQUALITY CONTROL TEST FOR cDNAThis test was being performed by PCR amplification of β-Actin.Reaction mixture was prepared with following componentscDNA - 1µL 53    
  • 54. 10XRT buffer - 1µL P.T.O2.5mMdNTP - 1µL20 uM forward primer - 0.09ul20 uM reverse primer - 0.09ulTaq polymerase - 0.2ulH 2O - 6.62ul 10ulPCR conditions95°`C  3 min95°C  30 sec56.7°C  15 sec72°C  30 sec72°C  10 min 4°C  ∞Following PCR,the product was run in 1% gel and analysed for bandscorresponding to B-actin. 54    
  • 55. Experiment B15 REAL-TIME PCRAimThis technique is used to amplify and simultaneously quantify atargeted DNA molecule. Here the amplified DNA is detected as the reactionprogresses in real time. Two common methods for detection of products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with anydouble-stranded DNA, and (2) sequence-specific DNA probes consistingof oligonucleotides that are labeled with a fluorescent reporter which permitsdetection only after hybridization of the probe with its complementary DNAtarget. 55    
  • 56. There are basically two methods of analyzing the data from a real time PCR-absolute quantification and relative quantification. Relative quantification orcomparative quantification measures the relative change in mRNA expressionlevels. It determines the changes in steady state mRNA levels of a gene acrossmultiple samples and expresses it relative to the levels of another RNA. Relativeconcentrations of DNA present during the exponential phase of the reaction aredetermined by plotting fluorescence against cycle number on a logarithmicscale (so that an exponentially-increasing quantity will show as a straight line).A threshold for detection of fluorescence above background is determined. Thecycle at which the fluorescence from a sample crosses the threshold is calledthe cycle threshold, Ct.The amount of target, normalized to an endogenous reference and relative to acalibrator is given by, Amount of target = 2-ΔΔCTWhere 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 thetarget gene normalized to β-actin.ProcedurePCR components for TaqMan based gene expression assaycDNA - 2µlTaqMan Universal PCR Master Mix - 5 µl20X TaqMan Gene Expression Assay Mix - 0.5 µlNuclease-free water - 2.5 µlTotal - 10 µl 56    
  • 57. Reaction mix was prepared for B-actin also, which served as the referencegene.The samples were loaded on 96-well plate and each sample was run intriplicates.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 ofStep 3.15 sec at 95 C and then 1 minute at 60 C.The results were analyzed using SDS RQ Manager software.. 57    
  • 58. Reference:Section AMolecular  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 Bhttp://www.research.umbc.edu/~jwolf/method1.htm  Sigma-Aldrich ECACC handbookhttp://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