User Manual
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1. Manual hot start is not required B PCR protocol 1 Prepare a PCR master mix by combining the following reagents in the order shown in Table 1 2 Mix by vortexing without bubbling and spin the tube briefly in a microcentrifuge 3 Immediately before thermal cycling aliquot the PCR master mix into an appropriate number of sterile 0 2 ml or 0 5 ml PCR tubes Note Thin wall PCR tubes are recommended These PCR tubes are optimized to ensure more efficient heat transfer and to maximize thermal cycling performance 4 f required add missing components i e components that vary from one reaction to the other 5 f your thermal cycler is not equipped with a heated cover overlay each reaction with a drop of molecular biology grade mineral oil 6 Perform PCR using the optimized cycling conditions as described in the section III D 7 f required analyze the PCR amplification products using electrophoresis on a 1 0 4 0 w v agarose gel with etidium bromide EtBr staining Recommendations related to agarose gel electrophoresis are available in the Appendix A PCR products can be stored at 20 C technical support customer support evrogen com 4 Table 1 Setting up PCR master mix Component Final reaction volume 25 ul 50 ul concentration Sterile water addto25ul addto50ul 10X Encyclo buffer 2 5 ul 5 ul 1X 50X dNTP mix 0 5 ul 1 yl 1X 0 2 mM each Upstream Primer variable variable 0 2 0 5 uM Downstream Pri2. be performed on DNA or cDNA prepared by any common method as well as on cell lysates or bacterial colonies However template quality is strictly important for amplification of long templates e g templates longer than 3 kb or when highest possible sensitivity of PCR is needed For example such PCR applications as RACE and Genome Walking require high quality templates Please note that a number of compounds can inhibit PCR amplification including ionic detergents some gel loading dyes ethanol phenol and hemin When purifying templates from agarose gels minimize exposure to UV irradiation to prevent formation of pyrimidine dimers The amount of DNA template required on PCR start varies depending on the source quality and length of DNA being amplified In many applications the optimal amount of a low complexity template cloned DNA fragments lambda DNA etc ranges between 1 pg 10 ng per 50 ul reaction 20 50 ng per 50 ul reaction for cDNA and 50 200 ng per 50 ul reaction for high complexity genomes e g human genomic DNA Excessive amounts of template DNA can inhibit PCR 3 Primers Primer concentrations between 0 2 and 0 5 uM are recommended generally 100 250 ng for typical 20 to 30 mer oligonucleotide primers in a 50 yl reaction volume Primers designed to have similar melting temperatures are recommended Primers with melting temperatures less than 50 C are not recommended for use with Encyclo PCR kit The anneali
3. EVRQOLGEN Innovative Biotechnology Company Evrogen JCS Miklukho Maklaya str 16 10 117997 Moscow Russia Tel 7 495 429 8020 Fax 7 495 429 8520 www evrogen com Encyclo PCR kit Cat PKOO1 User Manual This product is intended for research use only TABLE OF CONTENTS Kit components and storage conditions Il Product description Ill Guidelines for PCR amplification A General Considerations B PCR protocol C Notes about Reaction Components D Cycling conditions E Control PCR IV Troubleshooting Guide V References VI Appendix A Recommendations for Electrophoresis B Dealing with contamination Encyclo PCR kit N NOOU www evrogen ru I Kit components and storage conditions Component Amount 50X Encyclo polymerase mix 100 wl 10X Encyclo buffer 600 ul 50X dNTP mix 10mM each 120 ul Control DNA template 10 ul Control PCR primer mix 10 uM each primer 10 ul Sterile PCR water 1 5 ml Encyclo PCR kit provides reagents for 100 standard PCR reactions 50 ul each The kit includes 1 5 ml of PCR grade H O which is enough for approximately 40 standard PCRs We recommend to use Millipore filtered H O for the rest of reactions Do not use autoclaved H O for PCR Shipping Storage Encyclo Polymerase mix is shipped on dry ice or at 20 C All other components of Encyclo PCR kit can be shipped at ambient temperature Once arrived the kit must be kept at 20 C Il Product description Encyclo
4. PCR kit is intended for most PCR and primer extension applications It is especially recommended for cDNA amplification due to optimal combination of high fidelity and processivity provided by Encyclo polymerase mix Evrogen Encyclo polymerase mix produces high yields of PCR products from a wide variety of templates and is suitable for difficult templates long PCR up to 15 kb and cloning Encyclo polymerase mix features 1 High processive 5 gt 3 DNA polymerase activity 2 Proofreading 3 gt 5 exonuclease activity 3 Automatic hot start 4 TA cloning compatibility technical support customer support evrogen com 2 Encyclo buffer has been developed to provide successful amplification of long DNA templates and is necessary for optimal perfrormance of the Encyclo polymerasy mix The kit also includes a mix of high purity deoxyribonucleotides sterile PCR water control DNA template and primer mix for positive control PCR Product Use Limitations Encyclo PCR kit is intended for research purposes only Ill Guidelines for PCR amplification A General Considerations 1 Avoid cross contamination Minute amounts of contaminating DNA can lead to nonspecific amplification even in the absence of an added DNA template We recommend setting up PCRs in a dedicated lab area separately from that used for DNA preparation or analysis of PCR products Use PCR pipette tips containing hydrophobic filters to minimize cross contaminati
5. a high annealing temperature preferably 65 68 C Whenever possible design primer pairs with similar Tm values When two primers have different Tm use the lowest one for PCR cycling 3 Extension Extension should be performed at 72 C for the most PCR applications A constant extension time can be used one minute per 1 3 1 5 kb of expected extension product To enhance TA cloning and achieve complete DNA extension it may be helpful to include an additional incubation step of 2 10 min at 72 C at the end of cycling 4 Number of PCR cycles We recommend using the marginally possible number of PCR cycles since overcycling may yield a nonspecific PCR product If necessary undercycling can be easily rectified by placing the reaction tube back into the thermal cycler for a few more cycles see Troubleshooting Guide technical support customer support evrogen com A number of PCR cycles required to produce a certain amount of PCR product e g 5 10 ng yl strongly depends on the initial number of target DNA molecules used for PCR amplification This dependence may be formulated as follows N 2 where N means a number of DNA molecules at the start of amplification and n a number of PCR cycles required to amplify the product to yield the concentration of 5 10 ng ul For example for a 1 kb long DNA molecule weight is about 1078 g the following rules are correct when optimal PCR conditions are used Template amo
6. evrogen com Increase the number of PCR cycles 3 5 additional cycles at a time Decrease the annealing temperature in increments of 2 4 C Optimize denaturation temperature by decreasing or increasing it in 1 C increments Increase the extension time in 1 min increments Redesign your primer s after confirming the accuracy of the sequence information If the original primer s was less than 22 nt long try using a longer primer s Repeat PCR varying the concentration of DNA template Check template integrity by agarose EtBr gel electrophoresis If necessary repurify your template using methods that minimize DNA nicking www evrogen com B Multiple PCR products or smear observed Template DNA may comprise components inhibiting PCR Template DNA may be difficult to PCR Too little enzyme Mg concentration is too low Too many cycles Annealing temperature too low Denaturation temperature too low Extension time too long Suboptimal primer Repurify your template Try to use DMSO additive in the concentration of 2 6 In rare cases the PCR yield can be improved by increasing the concentration of the enzyme mix However increasing the concentration gt 2X is likely to lead to higher background levels Encyclo polymerase has a broader Mg optimum than the native Taq DNA polymerase and can be used over a wider range of Mg without loss of efficiency Therefore as long as you
7. g PCR directly on phage plaques or bacterial colonies failure to isolate single plaques or colonies will also produce multiple bands Try a lower concentration of DNA template in the PCR reaction Check template integrity by agarose EtBr gel electrophoresis If necessary re purify your template If smearing is observed first try optimizing the cycle parameters as described above then try reducing the enzyme concentration to 0 5X Encyclo polymerase mix Touchdown PCR significantly improves the specificity of many PCR reactions in various applications Don et al 1991 Roux 1995 Touchdown PCR involves using an annealing extension temperature that is several degrees higher than the Tm of the primers during the initial PCR cycles The annealing extension temperature is then reduced to the primer Tm for the remaining PCR cycles The change can be performed either in a single step or in increments over several cycles Perform an additional PCR using nested of step out primers See detailed description of Step out PCR in Matz et al 1999 www evrogen com 13 V References 1 Chester N Marshak D R 1993 Dimethyl sulfoxide mediated primer Tm reduction a method for analyzing the role of renaturation temperature in the polymerase chain reaction Anal Biochem 209 2 284 290 2 Don R H Cox P T Wainwright B J Baker K amp Mattick J S 1991 Touchdown PCR to circumvent spurious priming during
8. gene amplification Nucleic Acids Res 19 4008 3 Matz M Shagin D Bogdanova E Britanova O Lukyanov S Diatchenko L Chenchik A 1999 Amplification of cDNA ends based on template switching effect and step out PCR Nucleic Acids Res 27 6 1558 1560 4 Roux K H 1995 Optimization and troubleshooting in PCR PCR Methods Appl 4 5185 5194 VI Appendix Appendix A Recommendations for Electrophoresis Transfer a 2 5 ul sample of your PCR reaction to a fresh tube and add 1 ul of 5X loading buffer The remaining reaction mixture can be subjected to further cycling if you do not see a product Analyze your sample s along with suitable DNA size markers by electrophoresis on a suitable agarose gel containing 0 1 ug ml EtBr The appropriate percentage of agarose and the choice of DNA size markers depend on the expected size range of a PCR product You may wish to refer to the following general guidelines before assembling your gel Expected insert size range Recommended agarose 0 3 1 5 kb 1 5 0 5 10kb 1 2 gt 5 kb 0 8 technical support customer support evrogen com 14 Appendix B Dealing with contamination If possible set up the PCR reaction and perform the post PCR analysis in separate laboratory areas with separate sets of pipettors It is advisable to use one of the commercially available aerosol free pipette tips Laboratory benches and pipettor shafts can be decontaminated by depurination Wipe s
9. mer variable variable 0 2 0 5 uM DNA template variable variable lpg 200ng 50 ul 50X Encyclo 0 5 ul 1 yl 1X polymerase mix Total volume 25 ul 50 ul The recipe is for one reaction and must be adjusted for multiple samples See section IIl C Notes about Reaction Components for more details on PCR components These components should be added into a PCR master mix when same components are used for all PCRs or into PCR tubes after PCR master mix aliquoting when different components are used in different PCRs C Notes about Reaction Components 1 Enzyme Buffer Mg2 concentration dNTP concentration The recommended amount of Encyclo polymerase mix allows successful PCR of DNA templates up to 15 kb Encyclo PCR buffer provided is essential for optimal yield and specificity of PCR Suboptimal results will be achieved using other buffers The provided 10X Encyclo buffer contains the magnesium ion concentration optimal for the Encyclo polymerase mix Adjusting the magnesium concentration is not recommended High quality dNTPs provided should be used for optimal performance with Encyclo polymerase mix Optimal dNTP concentration is 200 uM of each dNTP Adjusting the dNTP concentration is not recommended for templates up to 10 kb dUTP and other dUTP derivatives or analogues were not tested Encyclo PCR kit www evrogen com 2 Templates Template quality is not crucial for many conventional applications so that PCR may
10. mined Optimization of PCR parameters allows achieving highest product yield and specificity Table 2 PCR cycling parameters Cycle step Number Temperature Duration of cycles Initial denaturation 1 92 95 C 1 min 3 min Denaturation 92 95 C 5 sec 1 min Annealing 10 35 Tm 5 sec 1 min Extension 72 C 1 minute 1 1 5 kb Final extension Tm 5 sec 1 min optional 72 C 2 10 min Encyclo PCR kit www evrogen com 1 Denaturation Denaturation time and temperature depend at least in part on the ramp rate and temperature control mode of the thermal cycler After an initial denaturation for up to 3 min at 92 95 C keep the denaturation as short as possible for example 20 sec or less at 95 94 C This is particularly important for long PCR Initial denaturation for 3 min is recommended for complex genomic DNA while shorter time up to 2 min should be used with simpler templates 2 Primer annealing Optimal primer annealing temperature depends on the primer structure Typically annealing temperatures range between 55 and 72 C Simplified formula for estimating annealing temperature Tm is Tm 2 C x A T 4 C x G C Optimal annealing temperatures may be above or below the estimated Tm for up to 5 C In many cases use of an annealing temperature wich is 5 C above the calculated Tm i e Tm 5 C can sharply increase PCR specificity To achieve maximal reaction specificity use primers designed to have
11. ng temperature of primers 20 nt or more in length with a 45 60 GC content is generally between 60 and 72 C promoting their specificity and discouraging secondary structure formation Primer sequences should be analyzed for potential duplex and hairpin formation as well as false priming sites in order to obtain the highest yield of specific PCR products technical support customer support evrogen com 6 Avoid complementarity of two or three bases at the 3 ends of primer pairs to reduce primer dimer formation Avoid runs of 3 or more Gs or Cs at the 3 end Avoid complementary sequences within a primer sequence and between the primer pair 4 PCR additives Encyclo polymerase mix tolerates DMSO concentrations up to 6 needed to open up complex secondary structures within DNA templates We recommend using DMSO for difficult templates in concentrations of 2 6 Note In high DMSO concentrations the annealing temperature must be lowered because DMSO decreases the melting point of the primers It has been reported that 10 DMSO decreases the melting temperature by 5 5 6 0 C Other additives which help DNA denaturation formamide glycerol betaine and combinations of these have not been tested with Encyclo polymerase mix yet D Cycling conditions Use Table 2 to determine PCR cycling parameters Optimal cycling conditions such as incubation times temperatures and the number of cycles may vary and must be individually deter
12. of the control template generates a 1600 bp product Fig 1 3 0 2 0 1 5 1 0 Figure 1 Successful result of a control PCR Control PCR was performed as described in the section IIE PCR products lane 1 were visualized by electrophoresis on 1 5 agarose EtBr gel alongside of 1 kb DNA size markers lane M 50 ng per lane Sibenzyme technical support customer support evrogen com 10 IV Troubleshooting Guide The following general guidelines apply to most PCR reactions However no attempt has been made to address troubleshooting for all of the many applications for which the Encyclo PCR kit can be used Problem A Low yield or no product observed Encyclo PCR kit Putative causes PCR component missing or degraded Not enough PCR cycles Annealing temperature too high Denaturation temperature too high Extension time too short Suboptimal primer design Too high or too low template concentration Template DNA may be damaged Suggestions Use a checklist when assembling reactions Do not use buffers optimized for another polymerase Always perform a positive control to ensure that each component is functional If the positive control does not work repeat the positive control only If the positive control does not work again try optimizing PCR parameters for your particular thermal cycler If the positive control still does not work contact Evrogen technical support customer support
13. on It is strongly recommended to include a negative control in which sterile water is used instead of DNA template in every experiment 2 Include positive control Always perform a positive control to ensure that each component is functional See section III E Control PCR for details 3 Use PCR master mix Use of a PCR master mix reduces tube to tube variations in multiple PCR The master mix typically contains all components needed for PCR except for those varying from one reaction to another For example if multiple templates are being tested with the same primers include the primers in the master mix If one template is being tested with multiple primer sets include the template in the master mix Prepare a volume of master mix 10 greater than that required for the total number of PCR assays including positive and negative controls Encyclo PCR kit www evrogen ru The master mix should be thoroughly mixed before use 4 Use careful pipetting technique When small volumes of reagents are used in PCR experiments careful pipetting technique is crucial to avoid tube to tube variations Always be sure that no extra solution is on the outside of a pipette tip before transfer When adding solution to a tube immerse the tip into the reaction mixture deliver the solution and rinse the pipette tip by pipetting up and down several times 5 Do not use manual hot start Encyclo polymerase mix provides automatic hot start
14. unt at PCR Number of PCR cycles start 50 wl reaction to amplify 5 10 ng pl of DNA 1 molecule AO cycles 1000 molecules 30 cycles 10 molecules 1 pg 20 cycles 10 molecules 1 ng 10 cycles Please keep in mind that a PCR product visible on agarose EtBr gel only after 40 or more PCR cycles is amplified from a single molecule and could result from a casual contamination E Control PCR 1 Assemble the reaction using the Control Template and Control Primers Mix in a sterile PCR tube AO ul Sterile water 5 ul 10X Encyclo Buffer 1 ul 50X dNTP mix 2 ul Control Primer mix 1 ul Control DNA template 1 ul 50X Encyclo polymerase mix 50 ul Total volume Encyclo PCR kit www evrogen com 2 Mix gently and spin the tube briefly in a microcentrifuge 3 If the temperature cycler is not equipped with a heated cover overlay each reaction with two drops of mineral oil 4 Commence thermal cycling using the following parameters a Initially denature the template at 95 C for 2 minutes b Perform 18 cycles Denaturation 95 C 20 sec Annealing 60 C 20 sec Elongation 72 C 2 min c Perform a final extension at 72 C for 2 minutes PCR cycling parameters have been optimized for MJ Research PTC 200 DNA Thermal Cycler Optimal parameters may vary with different thermal cyclers polymerase lots and templates 5 Analyze the PCR amplification products by electrophoresis on a 1 0 4 0 w v agarose EtBr gel Amplification
15. urfaces with 1N HCI followed by 1N NaOH Then neutralize with a neutral buffer e g Tris HCl or PBS and rinse with ddH O Endnotes PCR is the subject of patents issued in certain countries The purchase of this product does not include a license to perform PCR However many researchers may not be required to obtain a license Other investigators may already have a license to perform PCR through use of a thermal cycler with the appropriate label license Material safety data sheet information EVROGEN JSC Moscow Russia hereby confirms that to the best of our knowledge this product does not require a Material Safety Data Sheet However all of the properties of this product and if applicable each of its components have not been thoroughly investigated Therefore we recommend that you use gloves and eye protection and wear a laboratory coat when working with this product Encyclo PCR kit www evrogen com Ver 01061
16. use the buffer included in the kit and a final concentration of 0 2 mM of each dNTP it is unlikely a lack of product is due to problems with the Mg concentration However if the concentration of EDTA in the cDNA sample is more than 5 mM this can reduce the effective concentration of Mg to below a minimum level Reducing the cycle number may eliminate nonspecific bands and smear Increase the annealing temperature in increments of 2 3 C Increase the denaturation temperature in increments of 1 C Decrease the extension time in min increments Redesign your primer s after design confirming the accuracy of the sequence information If the original primer s was less than 22 nt long try using a longer primer If the original primer s had a GC content of less than 45 try to design a primer with a GC content of 45 60 technical support customer support evrogen com 12 Encyclo PCR kit Contamination Too much template Poor template Too much enzyme Touchdown PCR is required Step out and or nested PCR is required Contamination most often results in extra bands or smearing It is important to include a negative control i e a control using sterile water instead of the DNA template in every PCR experiment to determine if the PCR reagents pipettes or PCR reaction tubes are contaminated with previously amplified targets see Appendix B for Dealing with contamination Also when performin
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