Detection of SNPs Using Molecular Biology
Contents
1. 15 Copyright Biochemistry amp ComputerScience 10 Centrifuge at 8000 rpm for 1 min 11 Place the QIAamp Spin Column in a clean 2 ml collection tube provided and discard the tube containing the filtrate Copyright O Biochemistry ComputerScience 12 Carefully open the OlAamp Spin Column and add 500 ul Buffer AW1 without wetting the rim 14 Place the QIAamp Spin Column in a clean 2 ml collection tube provided and discard the collection tube containing the filtrate 17 Copyright Biochemistry amp ComputerScience 15 Carefully open the QIAamp Spin Column and add 500 ul Buffer AW2 without wetting the rim 17 Place the OIAamp Spin Column in a clean 1 5 ml microcentrifuge tube not provided Discard the collection tube containing the filtrate 18 Copyright Biochemistry amp ComputerScience 18 Carefully open the QIAamp Spin Column and add 200 ul Buffer AE or distilled 19 Incubate at room temperature 15 25 C for 1 min incubating the QIAamp Spin Column loaded with Buffer AE or water for 5 min at room temperature before centrifugation generally increases DNA yield A second elution step with a further 200 ul Buffer AE will increase yields by up to 15 19 Copyright Biochemistry amp ComputerScience 20 Centrifuge at 8000 rpm for min 21 For long term storage of DNA eluting in Buffer AE and storing at 20 C is Recommended since2. For45sec A Fort mn 7 See more details about using Mastercycler Mastercycler gradient instrument 4 Copyright Biochemistry amp ComputerScience Cutting DNA fragments using restriction enzymes Restriction Enzymes recognize short DNA sequences They cleave double stranded DNA at specific sites within or adjacent to these sequence 160 Polymorphism of XRCCI will be determined by using Polymerase Chain Reaction Restriction Fragment Length Polymorphism PCR RFLP technique Amplified fragments will be digested by Msp I enzyme The PCR products were then subjected to restriction digestion overnight at 37 C with restriction enzymes 17 Recognition Sequence 5 CCG G 3 3 G GC a 48 Copyright Biochemistry amp ComputerScience Captured photos for Cutting DNA fragments using restriction enzymes Figure 5 2 Gel Electrophoresis X Restriction Y Enzymes d 1 Restriction enzymes cleave DNA into smaller segments of various sizes 2 DNA segments are loaded into wells in a porous gel The gel floats in a buffer solution within a chamber between two electrodes 3 When an electric current is passed through the chamber DNA fragments move toward the positively charged cathode 4 smaller DNA segments move faster and farther than larger DNA segments 18 Equipment Micropipettes 0 5 10 ul amp 20 200 ul PCR product tube Microcentrifuge tubes 1 5 ml
3. My tube contains 50 0 nmoles of oligo but I need a final oligonucleotide concentration of 1 0 uM in my reaction My reaction volume is 25 ul How much oligo do I add to my reaction First make a concentrated oligonucleotide stock solution 10X or 100X depending on the concentration you need Then add an appropriate volume to the reaction thereby achieving the desired working concentration Serial dilutions of the stock s may be required to ensure pipetting accuracy See outlined example below 1 Decide on a stock concentration and standardize your units Desired final concentration 1 00 uM 100X stock concentration 100 X 1 00 uM 100 micromole liter Total nanomoles of oligo 50 nmole see tube label for your actual oligo 33 Copyright Biochemistry amp ComputerScience e Total micromoles of oligo 30 1000 0 05 micromole Hint The quantity of oligo must be expressed in micromoles to calculate the volume for a micromolar solution 2 Make a 100X stock solution 100 X 1 00 uM 100 uM What volume V is required to resuspend 0 05 micromole of oligo to give a stock concentration of 100 uM Use the following steps as a guide 100 micromole 0 05 micromole Solve for V 1000 ml V ml V 0 05 X 1000 ml 0 5 ml 500 ul 100 3 Use serial dilutions to make a 10X stock 10 X 1 00 uM 10 uM For pipetting accuracy avoid using small volumes less than 2 ul of stock solutions in your reactions It may be n
4. Blood Mini Handbook 9 Copyright Biochemistry amp ComputerScience Captured photos for Genomic DNA extraction from whole blood 1 Number microcentrifuge tubes according to your samples E 10 Copyright Biochemistry amp ComputerScience 2 Pipet 20 ul QIAGEN Protease or Proteinase K into the bottom of a 1 5 ml microcentrifuge tube 11 Copyright Biochemistry amp ComputerScience 4 Add 200 ul Buffer AL to the sample Mix by pulse vortexing for 15 s Immediately then spin Hint in order to ensure efficient lysis it is essential that the sample and Buffer AL are mixed thoroughly to yield a homogeneous solution 12 Copyright O Biochemistry ComputerScience 5 Incubate at 56 C for 10 min Hint DNA yield reaches a maximum after lysis for 10 min at 56 C Longer incubation times have no effect on yield or quality of the purified DNA 13 Copyright O Biochemistry ComputerScience 6 Briefly centrifuge 1 5 ml microcentrifuge tube to remove drops from the inside of the lid 7 Add 200 ul of ethanol 96 100 to the sample and mix again by pulse vortexing for 15 s 14 Copyright Biochemistry amp ComputerScience 8 Briefly centrifuge 1 5 ml microcentrifuge tube to remove drops from the inside of the lid 9 Carefully apply the mixture from step 6 to the QIAamp Spin Column in a 2 ml collection tube without wetting the rim close the cap
5. and contamination because PCR amplification failure under optimum conditions for example primer dimer bands non specific bands smear and residues in wells DNA ladder Interested hand Primer dimer Loading dye Non specific band Interested band 67 Copyright O Biochemistry ComputerScience Residues in well The interested band 68 Copyright Biochemistry amp ComputerScience 13 Combine 2 ul of 6X loading buffer with 20 ul of restriction enzyme and then the digestion products is separated on agarose gel electrophoresis for identification DNA Ladder DNA Ladder vor ee Wild type 241 bp amp 374 bp PCR product 615 bp 69 Copyright Biochemistry amp ComputerScience Interpretation of data using statistical analysis Here you can find some statistical tests for interpretation of data 2 Two side x It was used to compare the distribution of the genotypes between cases and controls 20 Ca at at a lat Bet l bla Hardy Weinberg equilibrium It was tested by a goodness of fit x test to compare the observed genotype frequencies within the case control groups to the anticipated genotype frequencies calculated from the observed allele frequencies 20 2 lt Qi E x ZE i l Odds ratio This is a way of comparing whether the probability of a certain event is the same for two groups An odds ratio of implies that the event is equally l
6. electrode that will have the negative current 62 Copyright Biochemistry ComputerScience 9 To prepare samples for electrophoresis combine 2 ul of 6X loading buffer with 5 ul of PCR product and then mix well Hint PCR product is loaded onto 2 agarose gel to ensure that the amplification had occurred First method Transfer 5 ul from PCR product to Microcentrifuge tube 63 Copyright Biochemistry amp ComputerScience Throw a tip and take a new tip Combine 2 ul of 6X loading dye with 5 ul of PCR product in a microcentrifuge tube Transfer 5 ul of PCR product to a parafilm and combine it with 2 ul of 6X loading DNA Ladder Load samples into wells 64 Copyright Biochemistry amp ComputerScience DNA Ladder PCR products from different samples 10 The power supply can be monitored and operated in current amps voltage 100 volts in 30 minutes or power watts mode The black and red cords leading from the power supply are then attached to the tray in which the gel is run 65 Copyright Biochemistry amp ComputerScience 11 Put gel on UV transilluminator Pentax Optio MX4 Digital Camera is used to capture photo DNA Ladder 615 bp Different sizes of PCR Products from different samples 66 ea DNA Ladder Copyright Biochemistry amp ComputerScience 12 Troubleshooting in PCR sometimes using PCR can lead to undesirable bands
7. 8 C or 20 C When using the QIAamp DNA Blood Mini Kit 50 pipet 1 2 ml protease solvent into the vial containing lyophilized QIAGEN Protease as indicated on the label When using the OlAamp DNA Blood Mini Kit 250 pipet 5 5 ml protease solvent into the vial containing lyophilized QIAGEN Protease as indicated on the label Dissolved QIAGEN Protease is stable for up to 2 months when stored at 2 8 C Storage at 20 C is recommended to prolong the life of QIAGEN Protease but repeated freezing and thawing should be avoided For this reason storage of aliquots of QIAGEN Protease is recommended Buffer ALt store at room temperature 15 25 C Mix Buffer AL thoroughly by shaking before use Buffer AL is stable for 1 year when stored at room temperature Hint Do not add QIAGEN Protease or proteinase K directly to Buffer AL Buffer AW 1 store at room temperature 15 25 C Buffer AW 1 is supplied as a concentrate Before using for the first time add the appropriate amount of ethanol 96 100 as indicated on the bottle Buffer AWI is stable for 1 year when stored closed at room temperature 6 Buffer AW2 store at room temperature 15 25 C Buffer AW2 is supplied as a concentrate Before using for the first time add the appropriate amount of ethanol 96 100 to Buffer AW2 concentrate as indicated on the bottle Buffer AW2 is stable for 1 year when stored closed at room temperature Source QIAamp DNA Mini and
8. DNA stored in water is subject to acid hydrolysis Yield ug Sample Elution 1 Elution 2 Elution 3 Total Whole blood 20041 34 cn DNA concentration Elution volume Yield Yield ng ul 20 Copyright Biochemistry amp ComputerScience Determination of DNA concentration GeneQuant instrument is used to calculate the DNA concentration and its purity Equipment and Reagents 1 Distill water 2 DNA sample 3 Pathlength cell Procedure 1 For preparation of sample add 20 uL of DNA sample and complete with 1980 of D W in a pathlength cell 2 For preparation of blank add only 2000 ul of D W in a new pathlength cell 3 Read the blank and then measure the sample Source GeneQuant RNA DNA Calculator User Manual 21 Copyright Biochemistry amp ComputerScience Captured photos for calculation DNA concentration using GeneQuant instrument 22 Copyright Biochemistry amp ComputerScience Reference measurement 25 Copyright Biochemistry amp ComputerScience 24 Copyright Biochemistry amp ComputerScience Sample measurement 25 Copyright Biochemistry amp ComputerScience Amplification of DNA fragments by polymerase chain reaction What is PCR Polymerase Chain Reaction PCR means producing a large number of copies of a specific DNA sequence using a minimum amount of information about that sequence It is a technique widely used in the molecular bio
9. Detection of SNPs Using Molecular Biology Techniques By W Alghazzawi Copyright O Biochemistry amp ComputerScience Table of Contents OO A EE A ee 3 A O O O E E E E rece iets 4 Serum plasma or whole blood collection 6 Genomic DNA extraction from whole blood 8 Captured photos for genomic DNA extraction from whole blood 10 Determination of DNA concentration Rs 21 Captured Photos for determination of DNA concentration 22 Amplification of DNA fragments by polymerase chain reacti0n 26 Captured Photos for amplification of DNA fragments by polymerase chain A CUO DN aia 36 Cutting DNA fragments using restriction ENZYMe 48 Captured Photos for cutting DNA fragments using restriction enzyme 49 Running samples using agarose gel electrophoresis eee 56 Captured Photos for running samples using agarose gel electrophoresis 57 Interpretation of data using statistical analysis 70 Useful links for statistical analysis 72 OS O A 74 RN A O O A I 76 11 Copyright O Biochemistry ComputerScience Introduction This research focuses on the different techniques are used in the molecular biochemistry to detect Single Nucleotide Polymorphisms of human DNA These techniques organize sequential and include a collection of Blood plasma serum and whole blood DNA extraction from whole blood calculation of DNA concentration amplification of DNA fragments by polym
10. Reverse primer 5 TCC AGC CTT TTC TGA TA 3 4 Genomic DNA Preparation the stock primers 1 Always take great care not to contaminate the original primer stock 2 Use filter tips and PCR quality reagents 3 When you receive the lyophilized primer before opening it be sure to spin it down to insure that the primer pellet is at the bottom of the tube about 10000 rpm for 30 s 4 This tube will be used to make working primer solutions as needed and will be stored at 20 C or below 38 Copyright Biochemistry amp ComputerScience 5 Determine how many nmoles of primer is in the tube usually written on the tube or on information accompanying primer order 6 Resuspend the pellet in RNase free water that come with HotStar Tag Master Mix and mix well to generate the 100 uM stock solution An easy way to do this 1s to multiply the nmole value by 10 and add that volume in ul to the pellet For example If primer pellet is 5 nm you will add 50 ul RNase free water that come with HotStar Tag Master Mix to the pellet to resuspend it The final concentration of primer is 100 uM 7 Solutions should be aliquots in small portions and store Primer solutions at 20 C 8 39 Copyright Biochemistry amp ComputerScience Procedure HotStarTag Master Mix 12 5 ul RNase free water 10 1 ul Forward stock primer 0 1uM 0 2 ul Reverse stock primer 0 1uM 0 2 ul Genomic DNA 2 0 ul Total 25 ul Aliguot from F
11. S http www psychstat missouristate edu introbook SBK28 htm 9 HARDY WEINBERG EQUILIBRIUM http www tiem utk edu bioed bealsmodules hardy weinberg html 10 The Hardy Weinberg Law Animation http bcs whfreeman com piercele pages bcs main body asp s 23000 amp n 00020 amp 1 23020 01 amp v chapter amp 0o 1000 10100020100030100060 amp ns 0 amp t amp uid 0 amp rau 0 11 POPULATION GENETICS AND THE HARDY WEINBERG LAW http www k state edu parasitology biology198 hardwein html 12 Handbook of Biological Statistics http udel edu mcdonald statprobability html 73 Copyright Biochemistry amp ComputerScience Tables The below table is recommended to use it in your experiment for the results after runing PCR products on a gel and and the second table for the results after agter digestion of amplified fragments by restriction enzyme on a gel Name of PCR temperature It is recorded in a thermocycler instrument 74 Copyright Biochemistry amp ComputerScience 75 Copyright Biochemistry amp ComputerScience References 1 http www espionageinto com Pa Po Polymerase Chain Reaction PCR html 2 http en wikipedia org wiki Taq_polymerase 3 http en wikipedia org wiki Thermophilic 4 http en wikipedia org wiki Thermus_aquaticus 5 http en wikipedia org wiki Polymerase chain reactionfReferencesfReferences 6 HotStarTaq PCR Handbook 7 Mastercycler Mastercycler gradient man
12. Yellow tips Gloves Ice bucket LS A A SY E Reaction tube holder 49 Copyright O Biochemistry ComputerScience Reagents I se gt PCR product Msp I restriction enzyme RE 10X buffer Acetylated Bovine Serum Albumin BSA Injection water Procedure I Add 4 ul of PCR product to a new microcentrifuge tube 50 Copyright Biochemistry amp ComputerScience 2 Add 2 0 ul of RE 10X buffer to the reaction mixture using a new tip 51 Copyright Biochemistry ComputerScience 3 Add 0 5 ul of BSA to the same tube using a new tip 52 Copyright Biochemistry amp ComputerScience 53 Copyright Biochemistry amp ComputerScience 6 Spin tubes Total volume of reaction is 20 ul 7 Incubate the tubes at 37 C for overnight 19 54 Copyright Biochemistry amp ComputerScience Useful conversions 1 gram g 1000 milligrams mg I milligram mg 1000 microgram ug I microgram ug 1000 nanogram ng I nanogram ng 1000 picogram pg I gram 10e12 picograms 1 mole 1000 millimoles mmole 1 millimole mmole 1000 micromoles umole 1 micromole umole 1000 nanomoles nmole 1 nanomole nmole 1000 picomoles pmole I mole 10e12 picomoles A 1 micromolar solution 1uM 1 picomoles per microliter 1 pmoles ul luM 10e 6 moles liter x 10 6 liters ul 10e 12 moles ul 1 pmole ul 10 55 Copyright Biochemistr
13. al cycler and start the cycling program Hint After amplification samples can be stored overnight at 2 8 C or at 20 C Gradient PCR for Mastercycler gradient only The Gradient PCR is used to optimize temperatures in a PCR experiment The gradient may be programmed with a temperature range of up to 20 C with every temperature command The most common application is the determination of the optimal annealing temperature see example for which a gradient of for example 10 C is built up The recommended mean gradient is 5 C higher than the calculated annealing temperature 7 Gradient PCR Temperature control of tubes Temperature control of lid 1 Initial denaturation Denaturation Annealing Gradient Elongation Number of cycles Final elongation Cooling and storage End Y n E Control lid Nowa It gae gae 60 G 10 2 go to 12 Hold 20 end tube 105 auto 2 min 15 sec 15 sec 30 sec Rep 29 2 min enter Copyright Biochemistry amp ComputerScience e When the gradient is set at 61 C 10 C when 12 x 0 2 ml test tubes are used the temperature distribution in the individual columns of the thermoclock is as follows 7 ww 1 2 3 4 s e 7 e 9 w ul e When 0 5 ml test tubes are used the 11 rows of the block have the following temperatures CAC 2 3 4 s e 7 A wipn Calculation for the stock primer preparation 100 uM
14. ecessary therefore to serially dilute your stock oligonucleotide solution Make a 10X stock by diluting your 100X stock as follows 1 10 dilution of 100X stock 10 ul of 100 uM 100X stock 90 ul sterile water or TE 100 ul of 10 uM 10X stock 4 Use a 10X stock to give the final concentration in the reaction 9 A My reaction volume is 25 ul How much of the 10X 10 uM stock solution of oligo is required to yield a 1 00 uM concentration of oligo in the total reaction 34 Copyright Biochemistry amp ComputerScience Vol of 10X stock X stock conc Final reaction vol X final oligo conc S ul X 10 uM 23 ul X 1 0 uM Solve for S S 25 X10 2 5 ul 10 35 Copyright O Biochemistry ComputerScience Captured photos for Amplification of DNA fragments by polymerase chain reaction Equipment I A A A A lu Micropipettes 0 5 10 ul amp 20 200 ul PCR reaction tubes Microcentrifuge tubes 1 5 ml Yellow tips 0 200 ul Gloves Ice bucket Reaction tube holder PCR Tubes Thermocycler instrument Types of PCR tubes 36 Copyright Biochemistry amp ComputerScience The thermoblock has 96 narrow positions for 0 2 ml test tubes 37 The thermoblock has 77 wide positions for 0 5 ml test tubes Copyright Biochemistry amp ComputerScience Reagents 1 Components of HotStarTag Master Mix kit 2 Forward primer 5 TGC TTT CTC TGT GTC CA 3 3
15. erase chain reaction cutting DNA fragments using restriction enzymes running samples using agarose gel electrophoresis and interpretation of data using statistical analysis This research is also supported by captured photos to help academicians and students understanding these techniques Copyright Biochemistry amp ComputerScience Sterilization Cover each container tightly with aluminum foil and then sterilize glassware using autoclave machine before start your experiment Copyright Biochemistry amp ComputerScience Pour distilled water into the reservoir through opening on top of the autoclave until it reaches the bottom of the base of the safety valves The water reaches to border Set the time to 15 min Copyright Biochemistry amp ComputerScience Serum preparation ll 2 3 Keep the serum samples for within two weeks in lt 20 C Keep at Serum plasma or whole blood collection Use red top tube that it does not contain anti coagulant Whole blood is directly drawn into a blood tube Let blood clot at room temperature for at least 30 minutes Immediately centrifuge the clotted blood at 2 000 to 3 000 rpm for 15 minutes It 1s better to repeat the centrifugation for 3 times pa q ET v e 4 i ll Transfer and store serum samples into microcentrifuge tubes Label the tube carefully and write clearly number of sample name o
16. f patient and date 70 C for longer storage Copyright Biochemistry amp ComputerScience Plasma preparation 1 Use violet top tube or green top tube it contains anti coagulant 2 Mix the blood with the anticoagulant immediately after drawing each sample 3 Slowly invert the blood tube eight times 4 Leave the tube at least for 30 minutes to 1 hour at the room temperature until the RBCs are precipitated into the bottom of the tube and then the plasma yellow fluid 1s collected from the top of the tube into microcentrifuge tubes 5 Label the tube carefully and write clearly number of sample name of patient and date 6 Keep the serum samples for within two weeks in lt 20 C Keep at ci 70 C for longer storage Copyright Biochemistry amp ComputerScience Genomic DNA extraction from whole blood QIAamp DNA Blood Mini Kits Catalog numbers 51104 and 51106 are used for DNA extraction from the different sources In this research it will focus on the genomic DNA extraction from whole blood Equipment 1 Microcentrifuge tubes 1 5 ml 2 Micropipettes 20 200 ul and 200 1000 ul 3 Sterile tips 0 200 ul and 50 1000 ul 4 Vortex 5 Water bath Reagen s 1 QIAamp DNA Blood Mini Kit 2 Ethanol 96 100 Copyright Biochemistry amp ComputerScience Protocol using QIAamp DNA Blood Mini Kit l 2 QIAGEN Protease stock solution store at 2
17. ience Reaction composition using HotStar Taq Master Mix Component Wal recection Final cone HetStarle Master Mix 25 pl 2 5 units HotbtarTaqg DNA Polymerose Ix PER Buffer 200 pM of each dNTP Diluted primer mix Primer Variable 0 1 0 5 pM Primer B Variable 0 1 0 5 pM Distilled water provided Variable Template DMA Template DNA added at step 4 Variabk lt 1 pg reaction Total volume 50 pl Contoins 7 3 mA MgCl e Program the thermal cycler according to the manufacturer s instructions Each PCR program must start with an initial heat activation step at 95 C for 15 min A typical PCR cycling program is outlined below For maximum yield and specificity temperatures and cycling times should be optimized for each new template target and primer pair 6 Addinonal comments Ini al ecfivation step 15 min HatStarTac DNA Polymerase is activated by this heating step J step cycling Denaturaticn 0 5 min Annealing 0 5 1 min 5 below T of pen see appendix page 24 Extension min For PCR products longer thon 1 kb use on extension time of approxi mately 1 min per kb DMA Number of cycles 25 35 See appendix page 25 Final extension 10 min 31 Copyright Biochemistry amp ComputerScience e If you are using thermal cyclers with a heated lid do not use mineral oil e Filling volume Tube type 0 2 ml plate 5 to 50 ul Tube type 0 5 ml thin 5 to 100 ul e Place PCR tubes in the therm
18. ikely in both groups An odds ratio greater than one implies that the event is more likely in the first group An odds ratio less than one implies that the event 1s less likely in the first group 21 Odds Ratio A B C D AxD B xC 70 Copyright Biochemistry amp ComputerScience Hint The crude odds ratio is the unadjusted odds ratio ORs may be adjusted for confounding factors using either stratification methods Mantel Haenszel ORs or logistic regression Relative risk A more direct measure comparing the probabilities in two groups is the relative risk which is also known as the risk ratio The relative risk is simply the ratio of the two conditional probabilities Like the odds ratio a relative risk equal to 1 implies that the the event is equally probable in both groups A relative risk greater than implies that the event is more likely in the first group A relative risk less than 1 implies that the event is less likely in the first group 21 The relative risk is simply the ratio of the two conditional probabilities x x Row Total afla b b a b 100 Y c c d d c d 100 The relative risk RR for the event X would be given by the following formula pale 5 era d We can also define the risk ratio for the event X It would be the ratio of the conditional probabilities in the second column 1 e ble pp Mate ESA dfle d 71 Copyright Biochemistry amp ComputerScience Useful link
19. logy 1 4th cycle wanted gene L Ist cycle template DNA A s J 4 copies R copies l copies 32 copies 2 68 billion copies Andy Vierstracte 1999 Component of the PCR reaction mixture Template DNA Heat stable or thermostable Taq polymerase Deoxynucleotide triphosphate dNTPs building blocks of DNA A C G T Cofactor MgCl Buffer solution dH O Two primers that flank both sides of the DNA to be amplified 26 Copyright Biochemistry amp ComputerScience Genomic DNA It is purified DNA from specific source which contains the region of the DNA fragment to be amplified Taq DNA polymerase It is a thermostable DNA polymerase which it was originally isolated from thermophilic bacterium Thermus aquaticu Which copies the region to be amplified 2 4 dNTPs Deoxynucleotide triphosphate dNTP mixes are aqueous solutions containing dATP dCTP dGTP and dTTP It used with DNA polymerase to build the new DNA Cofactor MgCb Mg Ions form complexes with dNTPs Primers and DNA template which needed for DNA polymerase activity 27 Copyright Biochemistry amp ComputerScience Buffer solution Which provide a suitable chemical environment for the DNA polymerase also keep the reaction at the proper pH Two primers To amplify the specific sequence requires the sequence flanking region It is oligonucleotide is usually short 15 30 bp 5 28 Copyright Biochemistry a
20. make a concentrated stock solution Prepare small aliquots of working solutions containing 10 pmol yl to avoid repeated thawing and nn Store all primer solutions at 20 C Primer quality can be checked on a denaturing polyacrylamide gel a single band should be seen 29 Copyright Biochemistry amp ComputerScience Principle of PCR 1 Denaturation the temperature holds between 94 C to 987C 94 C for 1 2 min and 96 C for 60 s 2 Annealing the temperature holds between 40 C to 68 C 3 Elongation or Extension temperature holds between 70 C to 80 C for 0 5 3 min 1kb of fragment take 1 min and 2kb of fragment take 2 min Protocol using HotStarTaq Master Mix This protocol serves only as a guideline for PCR amplification Optimal reaction conditions such as incubation times and temperatures and amount of template DNA may vary and need to be determined individually e HotStarTag Master Mix provides a final concentration of 1 5 mM MgCl in the final reaction mix which will produce satisfactory results in most cases However if a higher Mg concentration is required prepare a stock solution containing 25 mM MgCl e Set up reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis e Use disposable tips containing hydrophobic filters to minimize cross contamination e Thaw primer solutions and then mix well before use 6 30 Copyright Biochemistry amp ComputerSc
21. mp ComputerScience General guidelines for standard PCR primers 6 Length G C content T Sequence Concentration Storage 18 30 nucleotides A0 60 Simplified formula for estimating melting temperature 7 F 2Cx 4 T 4 C x G C Whenever possible design primer pairs with similar T values Optimal annealing temperatures may be above or below the estimated T As a starting point use an annealing temperature 5 C below T Avoid complementarity of two or three bases at the 3 ends of primer pairs to reduce primer dimer formation Avoid mismatches between the 3 end of the primer and the ta rgettemplate sequence Avoid runs of 3 or more G or C at the 3 end Avoid a 3 endT Primers with a T at the 3 end have a greater tolerance of mismatch Ayoid complementary seguences within a primer sequence and between the primer pair Commercially available computer software e g Primer Designer 1 0 Scientific Software 1990 Oligo Rychlik and Rhoads 1989 can be used for primer design Spectrophotometric conversion for primers 1 Ass unit 20 30 pg ml Mol ar conversions Primer length pmol pg 20 pmol amp mer 168 119 ng 20mer 152 132 ng 25mer 121 165 ng 3Omer 10 198 ng Use 0 1 0 5 pM of each primer in PCR For most applications a primer concentration of 0 2 pM will be sufficient Lyophilized primers should be dissolved in a small volume of distilled water or TE to
22. nto wells It is carries slight negative charges in neutral buffers and thus migrate in the same direction as the DNA during electrophoresis It was prepared as follow bromophenol blue 0 25 w v and sucrose 40 w v then complete the volume to 10 ml by distilled water Preparation of agarose gel solution 1 For preparation of agarose gel make 2 w v of agarose solution 59 Copyright Biochemistry amp ComputerScience 2 Boil the solution until completely dissolve 3 Let the solution cool down to about 60 C at room temperature 60 Copyright Biochemistry ComputerScience 4 Add 1 ul of ethidium bromide stock per 10 ml gel solution Hint some researchers prefer soaking the gel in an ethidium bromide solution after running or put directly in a buffer that is filled the tank 5 Stir the solution to disperse the ethidium bromide 6 Pour the melted agarose directly from the conical flask onto the casting deck If necessary using a glass or yellow tip carefully to remove any trapped air bubbles form the gel solution 61 Copyright Biochemistry amp ComputerScience 7 When the gel has completely cooled and solidified carefully remove the comb by pulling straight up on the comb holder from both sides o Put the gel together with the rack into a chamber filled with 1x TBE buffer Make sure the gel is completely covered with TBE buffer and that the slots are at the
23. orward Reverse primers RNase ada free water Genomic Genomic DNA Sample Genomic DNA Sample Stock Forward amp Reverse primers 1 Sterilize the hood using ethanol and wear gloves Copyright Biochemistry amp ComputerScience 2 Thaw all solutions before use 3 Mix the HotStarTaq Master Mix tube by vortexing briefly and dispense 12 5 ul into each PCR tube It 1s important to mix the HotStarTaq Master Mix before use in order to avoid localized concentrations of salt 3 Label PCR tubes by writing the number of sample 41 Copyright Biochemistry amp ComputerScience 4 Add 12 5 ul of HotStarTaq Master Mix to a clean PCR tube 42 Copyright Biochemistry ComputerScience 6 Add 10 1 ul of RNase free water to the same tube using a new tip r 43 Copyright Biochemistry amp ComputerScience 44 Copyright Biochemistry ComputerScience 9 Add DNA sample to the same tube using a new tip Total volume of reaction 1s 25 ul Copyright Biochemistry ComputerScience 10 Gently vortex the sample and briefly centrifuge for 10 min at 3000 rpm to collect all drops from walls of tube 11 All PCR tubes are placed in the mastercycler gradient thermocycler which can heat and cool the tubes 46 Copyright Biochemistry amp ComputerScience 12 Insert temperatures and then press START button to begin the reaction 4 T 72 C
24. rose will be small It means the smaller size of DNA will migrate e Ethidium bromide 10 mg ml in H20 is an intercalating Agent e Loading buffer 57 Copyright Biochemistry amp ComputerScience TBE buffer It is widely used for the electrophoresis of nucleic acids and has a higher buffer capacity than TAE It can be used for DNA and RNA polyacrylamide and agarose gel electrophoresis 5 Composition Atypical mixture of TBE consist of 108 g l 0 89 M EDTA Na2 salt 7 44 g l 0 02 M 55 0 g L 0 89 M Preparation 10X stock solution Tris Base Borate EDTA TBE buffer is containing 10X stock solutions are made as follows 108 gm of Tris base 55 gm of Boric acid and 7 44 gm of EDTA The ingredients were dissolved in 960 ml of distill water The pH is adjusted to 8 3 and the volume was complete to IL Preparation 1X dilute solution from 10X stock solution Take 100 ml from 10X stock solution and complete to 1000 ml with distill water Then the IX dilute solution is ready to fill in the gel tank to submerge in depth about 3 5 mm and to preparation agarose gel Preparation of Ethedium Bromide 10 mg ml Dissolve 0 1 gm of Ethedium Bromide in 10 ml of distill water 6 58 Copyright Biochemistry amp ComputerScience Preparation of 6X loading buffer The example of loading buffer IS Bromophenol blue tetrabromophenolsulfonephthalein It is used a color marker and density to the sample when load i
25. s for statistical analysis 1 Calculator online A For a 2x2 Contingency Table e Rates Risk Ratio Odds Odds Ratio Log Odds e Phi Coefficient of Association e Chi Square Test of Association e Fisher Exact Probability Test http faculty vassar edu lowry odds2x2 html B Calculate probability from X and d amp Calculate X from probability Q and d http www fourmilab ch rpkp experiments analysis chiCalc html C The Chi Square Statistic http math hws edu javamath ryan ChiSquare html D Chi Square Calculator http www pcrstation com E Simple Interactive Statistical Analysis http www quantitativeskills com sisa F QuickCalc http www graphpad com quickcalcs contingency1 cfm 2 ANSWERS POPULATION GENETICS PROBLEMS http science kennesaw edu rmatson B101 203380 ANS WERS html 12 Copyright Biochemistry amp ComputerScience 3 Critical Values of the Chi Square Distribution http www 1tl nist gov div898 handbook eda section3 eda3674 htm 4 The Chi Sguared Distribution Table http mips stanford edu public classes stats data analysis principles ch1 table html 5 Table of the Chi Square Distribution http www fmi uni sofia bg vesta virtual labs tables tables3 html 6 Critical Points of the Chi Sguare Distribution http www math unb ca knight utility chitable html 7 ON LINE CHI SQUARED TABLE http www unc edu farkouh usefull chi html 8 CHI SQUARE AND TESTS OF CONTINGENCY TABLE
26. ul 8 http www mbi ufl edu rowland protocols pcrprimer htm 9 http www sigmaaldrich com Brands Sigma_Genosys Custom_DNA Key_Re sources You_and_Your_Oligo html 10 http wheat pw usda gov lazo methods donis 16 16_5 html 11 http userpages umbc edu jwolf m6 htm 12 http www life utuc edu molbio geldigest electro html 13 http www vivo colostate edu hbooks genetics biotech gels agardna html 14 http mycoplasmas vm 1astate edu lab site methods DN A agarosegel html 15 Sambrook Fritsch Maniatis 1989 Molecular Cloning Cold Spring Harbor Laboratory Press B 23 p 6 7 16 http en wikipedia org wiki Restriction enzyme 17 http cebp aacrjournals org cgi content full 10 2 125 18 http www stanford edu group hopes sttools figureid html 19 Usage information of Msp Irestriction enzyme catalog R6401 Promega Corporation 20 http math hws edu javamath ryan ChiSquare html 21 http www childrensmercy org stats definitions or htm 76 Copyright Biochemistry amp ComputerScience The End T1 Copyright Biochemistry amp ComputerScience
27. y amp ComputerScience Running samples using agarose gel electrophoresis Agarose Agarose 1s purified from agar Purified agarose is in powdered form and insoluble in water or buffer at room temperature but it dissolve in boiling water Different purities of agarose are commercially available as are agaroses with different melting properties High purity low melt agarose is often used if the DNA is to be extracted from the gel 11 14 Agarose Gel Electrophoresis It is a method used in molecular biology to separate DNA strands by size and to estimate the size of the separated strands by comparison to known fragments DNA ladder This is achieved by pulling negatively charged DNA molecules through an agarose matrix with an electric field Shorter molecules move faster than longer ones 11 14 56 Copyright Biochemistry amp ComputerScience Captured photos for Running samples using agarose gel electrophoresis Equipment e HE 33 Mini horizontal submarine unit and was obtained from Hoefer Amersham Biosciences AB powered by an EPS 601 See figure below e Conical flask amp balance e Micropipette 0 5 10 ul amp 20 200 ul e Microcentrifuge tube tips comb and tap Reagents e PCR product samples e Tris Borate Na2EDTA TBE buffer solution is commonly used e DNA ladder DNA Marker or DNA Standard e Agarose 2 w v Hint If you use a high concentration of agarose the pores of aga
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