Home

TopTaq PCR Handbook

1. 34 TopTag PCR Handbook 06 2010 Appendix C Number of PCR Cycles A cycling program usually consists of between 25 and 35 cycles depending on the number of copies of the starting template Too many cycles do not necessarily lead to a higher yield of PCR product instead they may increase nonspecific background and decrease the yield of specific PCR product Table 15 provides a general guideline for choosing the number of cycles Table 15 General guidelines for choosing the number of PCR cycles Number of copies Human of starting genomic Number template 1 kb DNA E coli DNA DNA of cycles 10 100 0 01 0 11 fg 0 05 0 56pg 36 360 pg 40 45 100 1000 olim 056 556 pg 0 36 3 6ng 35 40 1x 10 5x 104 1 1 55 fg 5 56 278 pg 3 6 179 ng 30 35 25 x 0 gt 55 fg gt 278 pg gt 179 ng 25 35 Refer to Table 11 page 31 to calculate the number of molecules When starting with cDNA templates it is important to take into account the efficiency of reverse transcription in cDNA synthesis which is on average 10 30 Refers to single copy genes Appendix D RT PCR To perform PCR using RNA as a starting template the RNA must first be reverse transcribed into cDNA in a reverse transcriptase reaction RT reaction Failure of the subsequent PCR is often a result of the limitations of the RT reaction On average only 10 30 of the original RNA molecules are reverse transcribed into cDNA The expression level of the target RNA molecule
2. TopTaq PCR Handbook Toplaa DNA Polymerase Toplaa Master Mix Kit For standard and specialized end point PCR applications without the need for optimization QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in E Purification of DNA RNA and proteins M Nucleic acid and protein assays MH microRNA research and RNAi H Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com QIAGEN is a member of the Forest Stewardship Council FSC For the production of printed materials Papler aus ver antwortune ae including handbooks QIAGEN has a policy to select suppliers that comply with FSC standards for printing Fecr cop9007 processes and well managed forests Contents Kit Contents 4 Shipping and Storage 4 Product Use Limitations 5 Product Warranty and Satisfaction Guarantee 5 Technical Assistance 5 Safety Information 6 Product Specifications 6 Quality Control 7 Introduction 9 Equipment and Reagents to Be Supplied by User 13 Protocols M PCR Using TopTaq DNA Polymerase 14 M PCR Using TopTaq DNA Polymerase and Solution 18 E PCR Using TopTaq Master Mix Kit 23 Tr
3. If too little template is used primers may not be able to find their complementary sequences Too much template may lead to an increase in mispriming events As an initial guide spectrophotometric and molar conversion values for different nucleic acid templates are listed in Tables 10 and 11 respectively Table 10 Spectrophotometric conversions for nucleic acid templates 1 Aygo unit Concentration pg ml Double stranded DNA 50 Single stranded DNA 33 Single stranded RNA 40 t Absorbance at 260 nm 1 For further information see our guide Critical Factors for Successful PCR To obtain a copy visit the QIAGEN web site at www giagen com or call one of the QIAGEN Technical Service Departments or local distributors see back cover 30 TopTag PCR Handbook 06 2010 Table 11 Molar conversions for nucleic acid templates Nucleic acid Size pmol yg Molecules pg 1 kb DNA 1000 bp 1 52 9 1 x 10 pUC19 DNA 2686 bp 0 57 3 4x 10 pIZ18R DNA 2870 bp 0 54 3 2 x 10 pBluescript Il DNA 2961 bp 0152 Gall x IO Lambda DNA 48 502 bp 0 03 1 8 x T0 Average mRNA 1930 nt LO 1 0 x 10 Genomic DNA Escherichia coli 4 7 x 10 3 0 x 10 1 8 x 10 Drosophila melanogaster 1 4 x 10 iI x OP 6 6 x 10 Mus musculus mouse 2 7 x 10 5 7 x 107 3 4 x 10 Homo sapiens human G23 3 10 HE IG ecru Base pairs in haploid genome t For single copy genes Appendix B Primer Design Concentration and Storage Standard PCR pri
4. Note After amplification samples can be stored overnight at 2 8 C or at 20 C for longer storage 8 When using Coralload Concentrate the PCR reaction can be directly loaded onto an agarose gel without prior addition of a PCR loading buffer and gel tracking dyes Coralload Concentrate contains a gel loading reagent and two gel tracking dyes Refer to Table 3 for equivalent DNA migration distances at different agarose gel percentages 16 TopTag PCR Handbook 06 2010 Table 3 Migration distances of gel tracking dyes TAE TBE agarose gel Red dye Orange dye g 8 0 8 500 bp 270 bp 80 bp lt 10 bp a 1 0 300 bp 220 bp 40 bp lt 10 bp Su 1 5 250 bp 120 bp 20 bp lt 10 bp sg 2 0 100 bp 110 bp lt 10 bp lt 10 bp 3 0 50 bp 100 bp lt 10 bp lt 10 bp TopTaq PCR Handbook 06 2010 17 Protocol PCR Using TopTaq DNA Polymerase and Q Solution This protocol is designed for using Q Solution in PCR assays Q Solution changes the melting behavior of DNA and can be used for PCR systems that do not work well under standard conditions When using Q Solution the first time for a particular primer template pair always perform parallel reactions with and without Q Solution This recommendation should also be followed if another PCR additive such as DMSO was previously used for a particular primer template pair When using Q Solution depending on the individual PCR assay the following effects may be obse
5. Using the MinElute system PCR products can be purified in higher concentrations due to the low elution volumes needed in this system Gel loading reagent and tracking dyes are effectively removed with the QlAquick and MinElute systems For more information about QlAquick and MinElute products please call QIAGEN Technical Services or your local distributor see back cover or visit www giagen com TopTaq PCR Handbook 06 2010 37 Appendix G Control of Contamination It is extremely important to include at least one negative control that lacks the template nucleic acid in every PCR setup to detect possible contamination General physical precautions E Separate the working areas for setting up the PCR master mix and DNA handling including the addition of starting template PCR product analysis or plasmid preparation Ideally use separate rooms E Use a separate set of pipets for the PCR master mix Use of pipet tips with hydrophobic filters is strongly recommended E Prepare and freeze small aliquots of primer solutions and dNTP mix Use of fresh distilled water is strongly recommended M In case of contamination laboratory benches apparatus and pipets can be decontaminated by cleaning them with a 1 10 dilution of a commercial bleach solution Afterwards the benches and pipets should be rinsed with distilled water General chemical precautions HM PCR stock solutions can also be decontaminated using UV light This method is
6. laborious however and its efficiency is difficult to control and cannot be guaranteed We recommend storing solutions in small aliquots and using fresh aliquots for each PCR M Another approach to preventing amplification of contaminating DNA is to treat individual reaction mixtures with DNase or restriction enzymes that cut between the binding sites of the amplification primers used before adding the template DNA sample Most commercial bleach solutions are approximately 5 25 sodium hypochlorate t When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier 38 TopTag PCR Handbook 06 2010 Ordering Information Product Contents Cat no TopTaq DNA 250 units Toplaa DNA Polymerase 200203 Polymerase 250 10x PCR Buffer 10x Coralload Concentrate 5x Q Solution 25 mM MgCl TopTaq DNA 4 x 250 units Toplaa DNA 200205 Polymerase 1000 Polymerase 10x PCR Buffer 10x Coralload Concentrate 5x Q Solution 25 mM MgCl TopTaq DNA 5000 units TopTaq DNA Polymerase 200207 Polymerase 5000 10x PCR Buffer 10x Coralload Concentrate 5x Q Solution 25 mM MgCl TopTaq Master Mix Kit 250 3x 1 7 ml 2x TopTaq Master Mix 200403 containing 250 units TopTaq DNA Polymerase in total 1x 1 2 ml 10x Coralload Concentrate 3 x 1 9 ml RNase Free Water reagents provided
7. Buffer is used to ensure optimal PCR performance E Set up all 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 M When downstream applications require fluorescence or absorbance measurements addition of Coralload Concentrate is not recommended unless an intermediate purification of the PCR product e g using QlAquick or MinElute PCR Purification Kits will be performed Things to do before starting M If required prepare a dNTP mix containing 10 mM of each dNTP Store this mix in aliquots at 20 C High quality PCR grade dNTP mix 10 mM is available from QIAGEN cat no 201900 Procedure 1 Thaw dNTP mix and primer solutions at room temperature or on ice Remove TopTaq DNA Polymerase TopTaq PCR Buffer and Coralload Concentrate from storage at 4 C It is important to mix all the solutions especially the TopTaq PCR Buffer com pletely before use to avoid localized concentrations of salts 2 Prepare a master mix according to Table 1 page 15 For most primer template systems it is not necessary to keep reaction vessels on ice Due to the unique buffer formulation TopTaq DNA Polymerase reaction mix exhibits significantly reduced polymerase activity at room temperature The master mix contains all the components needed for PCR except the template DNA Prepare a volume of m
8. Japan For further information about RNeasy Oligotex and Omniscript products contact your local QIAGEN Technical Services or distributor see back cover or visit www qiagen com t Please refer to the manufacturer s instructions for the amount of enzyme required 36 TopTag PCR Handbook 06 2010 Appendix E Touchdown PCR Touchdown PCR uses a cycling program with varying annealing temperatures It is a useful method to increase the specificity of PCR The annealing temperature in the initial cycle should be 5 10 C above the Tn of the primers In subsequent cycles the annealing temperature is decreased in steps of 1 2 C cycle until a temperature is reached that is equal to or 2 5 below the T of the primers Touchdown PCR enhances the specificity of the initial primer template duplex formation and hence the specificity of the final PCR product To program your thermal cycler for touchdown PCR you should refer to the manufacturer s instructions Appendix F Purification of PCR Products After amplification the PCR sample contains a complex mixture of specific PCR product and residual reaction components such as primers unincorporated nucleotides enzyme s salts mineral oil and probably nonspecific amplification products Before the specific PCR product can be used in subsequent experiments it is often necessary to remove these contaminants The QlAquick system offers a quick and easy method for purifying the final PCR product
9. Polymerase and the TopTaa Master Mix Kit can also be stored at 20 C in a constant temperature freezer at least until the expiration date see the inside of the kit lid 4 TopTaq PCR Handbook 06 2010 Product Use Limitations Toplaa DNA Polymerase and Toplaa Master Mix Kit are intended for molecular biology applications These products are not intended for the diagnosis prevention or treatment of a disease Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse QIAGEN will replace it free of charge or refund the purchase price We reserve the right to change alter or modify any product to enhance its performance and design If a QIAGEN product does not meet your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish Separate conditions apply to QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information A copy of QIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your
10. a thermal cycler with a heated lid Problems with the thermal cycler TopTaq PCR Handbook 06 2010 Perform PCR with different final concentrations of Mg from 1 5 5 0 mM in 0 5 mM steps using a 25 mM MgCl solution When using TopTaq DNA Polymerase use 1 25 units per 50 pl reaction If necessary increase the amount of Toplag DNA Polymerase in 0 5 unit steps Increase the number of cycles in steps of 5 cycles see Appendix C page 35 Decrease annealing temperature in 2 C steps Annealing time should be between 30 and 60 s Difficulties in determining the optimal annealing temperature can be overcome in many cases by performing touchdown PCR see Appendix E page 37 Denaturation should be at 94 C for 30 to 60 s Ensure that the initial 3 minute 94 C incubation step is performed as described in step 6 of the PCR protocols pages 16 21 and 24 Increase the extension time in increments of 1 min Review primer design see Appendix B page 31 For RT PCR take into consideration the efficiency of the reverse transcriptase reaction which averages 10 30 The added volume of reverse transcriptase reaction should not exceed 10 of the final PCR volume see Appendix D page 35 When performing PCR in a thermal cycler with a heated lid do not overlay the PCR samples with mineral oil if the heated lid is switched on as this may decrease the yield of PCR product Check the power to the thermal cycler and th
11. always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier TopTaq PCR Handbook 06 2010 29 Appendix A Starting Template Both the quality and quantity of nucleic acid starting template affect PCR in particular the sensitivity and efficiency of amplification Quality of starting template Since PCR consists of multiple rounds of enzymatic reactions it is more sensitive to impurities such as proteins phenol chloroform salts ethanol EDTA and other chemical solvents than single step enzyme catalyzed processes QIAGEN offers a complete range of nucleic acid preparation systems ensuring the highestauality templates for PCR These include the QlAprep system for rapid plasmid purification the GlAamp and DNeasy systems for rapid purification of genomic DNA and viral nucleic acids and the RNeasy system for RNA preparation from a variety of sources For more information about QlAprep QlAamp DNeasy and RNeasy products contact one of our Technical Service Departments see back cover or visit www qiagen com productfinder Quantity of starting template The annealing efficiency of primer to template is an important factor in PCR Owing to the thermodynamic nature of the reaction the primer template ratio strongly influences the specificity and efficiency of PCR and should be optimized empirically
12. cycling conditions repeat not optimal the PCR using Q Solution Follow the protocol on page 18 d Primer concentration not optimal The primer concentration of 0 2 pM is or primers degraded suitable for most primer template systems with TopTaq DNA Polymerase However when primer concentration optimization is desired repeat the PCR with different primer concentrations from 0 1 0 5 pM of each primer in 0 1 pM steps In particular when performing highly sensitive PCR check for possible degradation of the primers on a denaturing polyacrylamide gel e Problems with starting template Check the concentration storage conditions and quality of the starting template see Appendix A page 30 If necessary make new serial dilutions of template nucleic acid from stock solutions Repeat the PCR using the new dilutions When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier 26 TopTag PCR Handbook 06 2010 Comments and suggestions g h k m n o Mg concentration not optimal Enzyme concentration too low Insufficient number of cycles Incorrect annealing temperature or time Incorrect denaturation temperature or time Extension time too short Primer design not optimal RT reaction error PCR overlaid with mineral oil when using
13. for 200 x 50 pl reactions Related products HotStarTaq Plus DNA Polymerase for highly specific hot start PCR without optimization HotStarTaq Plus DNA 250 units HofStarTaa Plus DNA 203603 Polymerase 250 UJ Polymerase 10x PCR Buffer 10x Coralload PCR Buffer 5x QSolution 25 mM MgCl Supplied in a single tube Contains 15 mM MgCl Contains 3 mM MgCl and 400 pM each dNTP Larger kit sizes available see www qiagen com TopTaq PCR Handbook 06 2010 39 Ordering Information Product Contents Cat no HotStarTaq Plus Master Mix Kit for fast and highly specific amplification HotStarTaq Plus Master 3 x 0 85 ml HotStarTaq Plus Master 203643 Mix Kit 250 Mix containing 250 units of HotStarTaq Plus DNA Polymerase total 1 x 0 55 ml Coralload Concentrate 2 x 1 9 ml RNase Free Water for 250 x 20 pl reactions HotStar HiFidelity Polymerase Kit for highly sensitive and reliable high fidelity hot start PCR HotStar HiFidelity 100 units HotStar HiFidelity DNA 202602 Polymerase Kit 100 U Polymerase 5x HotStar HiFidelity PCR Buffer inc dNTPs 5x QSolution 25 mM MgSO RNase Free Water QIAGEN Fast Cycling PCR Kit for rapid and highly specific PCR on any thermal cycler QIAGEN Fast Cycling 2 x 1 ml QIAGEN Fast Cycling PCR 203743 PCR Kit 200 Master Mix 10 x Coralload Dye Q Solution RNase Free Water suitable for 200 x 20 pl reactions dNTP Set and dNTP Mix PCR Grade for sensitive and repro
14. minimize cross contamination When downstream applications require fluorescence or absorbance measurements addition of Coralload Concentrate is not recommended unless an intermediate purification of the PCR product e g using QlAquick PCR Purification Kits or MinElute PCR Purification Kits will be performed Things to do before starting E If required prepare a dNTP mix containing 10 mM of each dNTP Store this mix in aliquots at 20 C High quality PCR grade dNTP mix 10 mM is available from QIAGEN cat no 201900 Procedure 1 Thaw dNTP mix and primer solutions at room temperature or on ice Remove TopTag DNA Polymerase TopTaq PCR Buffer Coralload Concentrate and Q Solution from storage at 4 C It is important to mix all the solutions especially TopTaq PCR Buffer completely before use to avoid localized concentrations of salts When using Q Solution additional MgCl is not usually required 2 Prepare a master mix at room temperature according to Table 4 page 20 For most primer template systems it is not necessary to keep reaction vessels on ice Due to the unique buffer formulation TopTaq DNA Polymerase reaction mix exhibits significantly reduced polymerase activity at room temperature The master mix contains all the components needed for PCR except the template DNA Prepare a volume of master mix 10 greater than that required for the total number of PCR assays to be performed A negative control without tem
15. purification of PCR products 100 bp to 10 kb QlAquick PCR Purification 50 QlAquick Spin Columns Buffers 28104 Kit 50 Collection Tubes 2 ml QlAquick Gel Extraction Kit for gel extraction or cleanup of DNA 70 bp to 10 kb from enzymatic reactions QlAquick Gel Extraction 50 QlAquick Spin Columns Buffers 28704 Kit 50 Collection Tubes 2 ml Larger kit sizes available see www giagen com TopTaq PCR Handbook 06 2010 Al Ordering Information Product Contents Cat no dNTP Set and dNTP Mix PCR Grade for sensitive and reproducible PCR and RT PCR dNTP Mix PCR Mix containing 10 mM each of dATP 201900 Grade 200 pl dCTP dGTP and dTTP 1 x 200 pl dNTP Set PCR Grade 100 mM each dATP dCTP dGTP 201912 4 x 100 pl dTTP for 1000 x 50 pl PCR reactions For up to date licensing information and productspecific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor 42 TopTaq PCR Handbook 06 2010 Trademarks QIAGEN QlAamp QlAprep QlAquick Coralload DNeasy HotStarTaq MinElute Oligotex Omniscript RNeasy TopTaq Q Solution QIAGEN Group pBluescript Stratagene Inc Registered names trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law Pur
16. MgCl 25 mM Conductivity pH sterility and performance in PCR are tested TopTaq Master Mix Kit PCR reproducibility assay PCR reproducibility and specificity are tested in parallel amplification reactions The reactions must yield a single specific product 8 TopTag PCR Handbook 06 2010 Introduction TopTaq DNA Polymerase has been developed by QIAGEN to provide highly reliable end point PCR with unrivalled ease of use Until now all PCR enzymes required storage at 20 C however due to the unique proprietary TopTaq Stabilizer contained in the enzyme storage buffer TopTaq DNA Polymerase and Toplaa Master Mix Kits are the first PCR kits that can be stored routinely at 4 C This results in significant time savings as thawing of reagents is not required Furthermore all components can be combined at room temperature eliminating the need for working on ice The unique buffer formulation and a single pre optimized protocol eliminate the need for optimization of experimental parameters for individual primer template systems The optional addition of Coralload Concentrate to the PCR reaction enables direct loading of the PCR products onto agarose gels without the need to add gel loading buffer saving time and resources The TopTaq Master Mix Kit offers all of the benefits of TopTaq DNA Polymerase combined with the advantage of a ready to use master mix With the TopTaq Master Mix Kit separate pipetting of individual components is not
17. The volume added should not exceed 10 of the final PCR volume see Appendix D page 35 5 When using a thermal cycler with a heated lid proceed directly to step 6 Otherwise overlay each reaction with approximately 50 pl mineral oil 20 TopTag PCR Handbook 06 2010 6 Program the thermal cycler according to the manufacturer s instructions The PCR cycling program outlined below has been pre optimized for most primer template systems However if an optimization of the annealing temperature is required please refer to Appendix B Page 31 Table 5 Pre optimized cycling protocol is suitable for most PCR systems If optimization of annealing temperature is required see Table12 page 32 Appendix B for more information Extension 1 min me For PCR products longer than 1 kb use an extension time of approximately 1 min per kb DNA p Additional comments 8 8 Initial es 3 min 94 C o z 3 step cycling g 2 Denaturation 30s 9A C lt S Annealing 30s 60 C The annealing temperature of 60 C S 3 Q Number of cycles 25 35 See Appendix C page 35 Final extension lOmin 72 C 7 Place the PCR tubes in the thermal cycler and start the cycling program Note After amplification samples can be stored overnight at 2 8 C or at 20 C for longer storage 8 When using Coralload Concentrate the PCR reaction can be directly loaded onto an agarose gel without prior addition of a PCR loading buffer an
18. arose gel without prior addition of a PCR loading buffer and gel tracking dyes Coralload PCR Buffer contains a gel loading reagent and gel tracking dyes Please refer to Table 9 below to identify the dyes according to migration distance and agarose gel percentage and type Table 9 Migration distances of gel tracking dyes TAE TBE agarose gel Red dye Orange dye 0 8 500 bp 270 bp 80 bp lt 10 bp 1 0 300 bp 220 bp 40 bp lt 10 bp 1 5 250 bp 120 bp 20 bp lt 10 bp 2 0 100 bp 110 bp lt 10 h klO ba 3 0 50 bp 100 bp lt 10 bp lt 10 bp TopTaq PCR Handbook 06 2010 25 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www giagen com FAQ FAQList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www qiagen com Comments and suggestions Little or no product a Pipetffing error or missing reagent Repeat the PCR Check the concentrations and storage conditions of reagents including primers and dNTP mix b Wrong PCR buffer The 10x TopTaq PCR Buffer provided in the kit is required for the optimal performance c PCR cycling conditions are Using the same
19. as the unique PCR buffer formulation prevents nonspecific DNA synthesis at room temperature 4 Add template DNA lt 1 pg reaction to the individual tubes containing the master mix For RT PCR add an aliquot from the reverse transcriptase reaction This should not exceed 10 of the final PCR volume see Appendix D page 35 TopTaq PCR Handbook 06 2010 15 5 When using a thermal cycler with a heated lid proceed directly to step 6 Otherwise overlay each reaction with approximately 50 pl mineral oil 6 Program the thermal cycler according to the manufacturer s instructions The PCR cycling program outlined below has been pre optimized for most primer template systems However if optimization of the annealing temperature is required please refer to Appendix B page 31 a Z a o e a Oo hd Polymerase Table 2 Pre optimized cycling protocol Additional comments Initial denaturation 3min 94 C 3 step cycling Denaturation 30 s ode Annealing 30 s 60 C The annealing temperature of 60 C is suitable for most PCR systems If optimization of annealing temperature is required see Table 12 page 32 Appendix B for more information Extension Tmin 72 C For PCR products longer than 1 kb use an extension time of approximately 1 min per kb DNA Number of cycles 25 35 See Appendix C page 35 Final extension 10 min 72 C 7 Place the PCR tubes in the thermal cycler and start the cycling program
20. aster mix 10 greater than that required for the total number of PCR assays to be performed A negative control without template DNA should always be included 14 TopTag PCR Handbook 06 2010 Note The Mg concentration of 15 mM provided by the supplied 10x TopTaq PCR Buffer will produce satisfactory results in most cases However in rare cases reactions may be improved by increasing the final Mg concentration see Troubleshooting Guide page 27 F Y SE 2 Table 1 Recommended reaction composition using TopTaq DNA Polymerase S U F Component Volume reaction Final concentration Master mix 10x TopTaq PCR Buffer 5 pl 1x dNTP mix 10 mM of each 1 pl 200 pM of each dNTP Optional 10x Coralload 5 pl 1x Concentrate Primer A Variable 0 1 0 5 pM 0 2 pM is suitable for most PCR systems Primer B Variable 0 1 0 5 pM 0 2 pM is suitable for most PCR systems TopTaq DNA Polymerase 0 25 pl 1 25 units reaction RNase free water Variable Template DNA Template DNA Variable lt 1 pg reaction Total volume 50 pl Note If smaller reaction volumes are used please reduce the amount of each component accordingly Contains 15 mM MgCl TopTaq DNA Polymerase should only be used in combination with 10x TopTaq PCR Buffer 3 Mix the master mix thoroughly and dispense appropriate volumes into PCR tubes Mix gently e g by pipetting the master mix up and down a few times It is not necessary to keep PCR tubes on ice
21. at the thermal cycler has been correctly programmed 27 Comments and suggestions Product is multi banded a PCR cycling conditions not optimal b Annealing temperature too low c Primer concentration not optimal or primers degraded d Primer design not optimal Product is smeared a Too much starting template b Carryover contamination Using the same cycling conditions repeat the PCR using Q Solution Follow the protocol on page 18 Increase annealing temperature in 2 C steps Annealing time should be between 30 and 60 s Difficulties in determining the optimal annealing temperature can be overcome in many cases by performing touchdown PCR see Appendix E page 37 Repeat the PCR with different primer concentrations from 0 1 0 5 pM of each primer in 0 1 pM steps In particular when performing highly sensitive PCR check for possible degradation of the primers on a denaturing polyacrylamide gel Review primer design see Appendix B page 31 Check the concentration and storage conditions of the starting template see Appendix A page 30 Make serial dilutions of template nucleic acid from stock solutions Perform PCR using these serial dilutions When re amplifying a PCR product start the re amplification round using 1 pl of a 1 in 103 104 dilution of the previous PCR If the negative control PCR without template DNA shows a PCR product or a smear exchange all reagents Use disposable pi
22. chase of this product is not accompanied by a license under patents owned by Roche Molecular Systems Inc and F Hoffmann La Roche Ltd No rights to realtime PCR or to any primers probes or pathogens are conveyed expressly by implication or by estoppel Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the TopTaq DNA Polymerase Kit and the TopTaq Master Mix Kit to the following terms 1 The TopTaq DNA Polymerase Kit and the TopTaq Master Mix Kit may be used solely in accordance with the TopTaq PCR Handbook and for use with components contained in the Kit only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the TopTaa PCR Handbook and additional protocols available at www qiagen com Other than expressly stated licenses QIAGEN makes no warranty that this Kit and or its use s do not infringe the rights of third parties This Kit and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in a
23. d gel tracking dyes Coralload Concentrate contains a gel loading reagent and two gel tracking dyes Refer to Table 6 below for equivalent DNA migration distances at different agarose gel percentages TopTaq PCR Handbook 06 2010 21 Table 6 Migration distances of gel tracking dyes TAE TBE agarose gel Red dye Orange dye 0 8 500 bp 270 bp 80 bp lt 10 bp 1 0 300 bp 220 bp 40 bp lt 10 bp 1 5 250 bp 120 bp 20 bp lt 10 bp 2 0 100 bp 110 bp lt 10 bp lt 10 bp 3 0 50 bp 100 bp lt 10 bp lt 10 bp o sy CG ge i 5 o N 5 o E e A lt Zz a o G 8 22 TopTaq PCR Handbook 06 2010 Protocol PCR Using TopTaq Master Mix Kit Important points before starting MH This protocol offers convenient pre optimized primer concentrations and an annealing temperature that works for most primer template systems It is recommended to start with these pre optimized values E Set up all reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis M Use disposable tips containing hydrophobic filters to minimize cross contamination E Coralload Concentrate is not recommended when downstream applications require fluorescence or absorbance measurements without an intermediate purification of the PCR product e g using QlAquick PCR Purification Kits or MinElute PCR Purification Kits Procedure 1 Thaw primer solutions and te
24. ducible PCR and RT PCR dNTP Mix PCR Grade Mix containing 10 mM each of 201900 200 pl dATP dCTP dGTP and dTTP 1 x 200 pl dNTP Set PCR Grade 100 mM each dATP dCTP dGTP 201912 4 x 100 pl dTTP for 1000 x 50 pl PCR reactions Larger kit sizes available see www giagen com t Contains 15 mM MgCl Contains Factor SB dNTPs and optimized concentration of MgSO 40 TopTaq PCR Handbook 06 2010 Ordering Information Product Contents Cat no OlAxcel System for effortless automated DNA fragment and RNA analysis QlAxcel System Capillary electrophoresis device 9001421 including computer and BioCalculator Analysis software 1 year warranty on parts and labor QlAxcel kits for fast high resolution capillary electrophoresis QlAxcel DNA High GlAxcel DNA High Resolution Gel 929002 Resolution Kit 1200 Cartridge Buffers Mineral Oil QX Intensity Calibration Marker 12 Tube Strips QlAxcel DNA Screening Kit QlAxcel DNA Screening Gel 929004 Cartridge Buffers Mineral Oil QX Intensity Calibration Marker 12 Tube Strips QlAxcel DNA Large GlAxcel DNA Large Fragment Gel 929006 Fragment Kit 600 Cartridge Buffers Mineral Oil QX Intensity Calibration Marker 12 Tube Strips MinElute PCR Purification Kit for purification of PCR products 70 bp to 4 kb in low elution volumes MinElute PCR Purification 50 MinElute Spin Columns Buffers 28004 Kit 50 Collection Tubes 2 ml QlAquick PCR Purification Kit for
25. e 3 end have a greater tolerance of mismatch M Avoid complementary sequences within a primer sequence and between the primer pair E Commercially available computer software e g Primer Designer 1 0 Scientific Software 1990 Oligo Rychlik and Rhoads 1989 can be used for primer design 32 TopTag PCR Handbook 06 2010 Concentration a Spectrophotometric conversion for primers 1 A20 unit 20 30 pg ml E Molar conversions Primer length pmol pg 20 pmol 18mer 168 119 ng 20mer 152 132 ng 25mer 121 165 ng 25mer 121 165 ng 30mer 101 198 ng M Use 0 1 0 5 pM of each primer in PCR For most applications a primer concentration of 0 2 pM will be sufficient Storage Lyophilized primers should be dissolved in a small volume of distilled water or TE to make a concentrated stock solution Prepare small aliquots of working solutions containing 10 pmol pl to avoid repeated thawing and freezing Store all primer solutions at 20 C Primer quality can be checked on a denaturing polyacrylamide gel a single band should be seen When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier Degenerate PCR primers Occasionally the exact nucleotide sequence of the targettemplate DNA will not be known for instance when it has been ded
26. echnical 080 000 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1 122 330 Technical 01 800 7742 639 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 1800 742 4362 Fax 65 6854 8184 Technical 1800 742 4368 Spain Orders 91 630 7050 Fax 911 630 5145 Technical 9 1 630 7050 Sweden Orders 020 790282 Fax 020 790582 s Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN aes Sample amp Assay Technologies
27. emixed solution for fast and highly specific hot start PCR amplification TopTaq PCR Handbook 06 2010 11 TopTaq Procedure No thawing of reagents r Reaction setup at room temperature Distribute Add primers and template Amplification 12 TopTaq PCR Handbook 06 2010 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier E RNase free water Reaction tubes Pipets and pipet tips aerosol resistant Thermal cycler Mineral oil only if the thermal cycler does not have a heated lid Primers should be purchased from an established oligonucleotide manufacturer such as Operon Biotechnologies www operon com Lyophilized primers should be dissolved in TE to provide a stock solution of 100 pM concentration should be checked by spectrophotometry Primer stock solutions should be stored in aliquots at 20 C TopTaq PCR Handbook 06 2010 13 Protocol PCR Using TopTaq DNA Polymerase Important points before starting E The protocol offers convenient pre optimized primer concentrations and an annealing temperature that works for most primer template systems It is recommended to start with these pre optimized values a Z a o nS a Oo hd Polymerase E Itis essential that the provided 10x TopTaa PCR
28. erase PCR Buffer with 3 mM MgCl and 400 pM each dNTP see quality control label inside kit lid for lotspecific values The amplification efficiency is tested in parallel amplification reactions and is indicated under Amp PCR reproducibility and specificity are tested in parallel amplification reactions The reactions must yield a single specific product Linearized plasmid DNA is incubated with TopTaq DNA Polymerase in PCR Buffer Exonuclease activity per unit of enzyme is indicated under Exo Plasmid DNA is incubated with TopTaq DNA Polymerase in PCR Buffer Endonuclease activity per unit of enzyme is indicated under Endo RNA is incubated with TopTaq DNA Polymerase in PCR Buffer RNase activity per unit of enzyme is indicated under RNase Toplaa DNA Polymerase is incubated in storage buffer Protease activity per unit of enzyme is indicated under Protease Assays are performed under standard PCR conditions without primers TopTaq DNA Polymerase and human genomic DNA purified with the QlAamp DNA Blood Mini Kit The absence of PCR product is indicated by No under Self priming Buffers and Reagents PCR Buffer 10x Conductivity pH sterility and performance in PCR are tested Coralload Concentrate 10x Conductivity pH sterility dye concentrations and performance in PCR are tested QSolution 5x Conductivity pH sterility and performance in PCR are tested
29. ge 18 Pre optimized protocol and cycling program To reduce the time and effort needed for the optimization of experimental parameters the protocol offers convenient pre optimized primer concentrations and an annealing temperature that has been shown to work for a large variety of primer template systems For optimal results and for your convenience it is recommended to start with these pre optimized values The PCR cycling program has also been pre optimized to work for most primer template systems For optimal results please follow the cycling protocol provided in this handbook 10 TopTag PCR Handbook 06 2010 Specificity and sensitivity With its balanced potassium and ammonium ions the unique PCR Buffer used in combination with TopTaq DNA Polymerase and TopTaq Master Mix Kit promotes specific primer template annealing and simultaneously reduces nonspecific annealing Maximum yields of specific products are obtained even when using low template amounts Downstream applications Toplaa DNA Polymerase and the TopTaq Master Mix Kit are ideally suited for a wide variety of applications For high fidelity PCR we recommend the HotStar HiFidelity Polymerase Kit for highly sensitive and reliable high fidelity PCR without optimization For hotstart PCR we recommend HotStarlaq Plus DNA Polymerase for maximum specificity without optimization requirements For even more convenience we offer HotStarTaq Plus Master Mix which contains a pr
30. local distributor see back cover or visit www qiagen com Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of QIAGEN products If you have any questions or experience any difficulties regarding Toplaa DNA Polymerase Kit TopTaq Master Mix Kit or QIAGEN products in general please do not hesitate to contact us QIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please see our Technical Support Center at www qiagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www giagen com TopTaq PCR Handbook 06 2010 5 Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDSs These are available online in convenient and compact PDF format at www qiagen com support MSDS aspx where y
31. mers Prerequisites for successful PCR include the design of optimal primer pairs the use of appropriate primer concentrations and the correct storage of primer solutions Some general guidelines are given in Table 12 For further information see our guide Critical Factors for Successful PCR To obtain a copy visit the QIAGEN web site at www giagen com or call one of the QIAGEN Technical Service Departments or local distributors see back cover TopTaq PCR Handbook 06 2010 31 Table 12 General guidelines for standard PCR primers Length 18 30 nucleotides G C content 40 60 T Simplified formula for estimating melting temperature Ta Ty 2 C x AT 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 If the recommended annealing temperature of 60 C mentioned in the protocol on pages 16 21 and 24 does not give satisfactory results please carry out a temperature gradient PCR with annealing temperatures from 50 68 C to identify the optimum annealing temperature Sequence M Avoid complementarity of two or three bases at the 3 ends of primer pairs to reduce primer dimer formation M Avoid mismatches between the 3 end of the primer and the targeHemplate sequence M Avoid runs of 3 or more G or C at the 3 end E Avoid a 3 end T Primers with a T at th
32. mplate DNA Mix well before use 2 Mix the TopTaq Master Mix by vortexing briefly and dispense 25 pl into each PCR tube according to Table 7 It is important to mix the TopTaq Master Mix before use in order to avoid localized concentrations of salt For most primer template systems it is not necessary to keep reaction vessels on ice since TopTaq Master Mix exhibits significantly reduced polymerase activity at room temperature due to the unique buffer formulation 3 Distribute the appropriate volume of diluted primer mix into the PCR tubes containing the Master Mix 4 Add template DNA 1 pg reaction to the individual PCR tubes For RT PCR add an aliquot from the reverse transcriptase reaction The volume added should not exceed 10 of the final PCR volume see Appendix D page 35 TopTaq PCR Handbook 06 2010 23 oH 8 fe a fe s 1 Xx Table 7 Recommended reaction composition using TopTaq Master Mix Component Volume reaction Final concentration TopTaq Master Mix 2x 25 pl 1 25 units TopTaq DNA Polymerase 1 x PCR Buffer 200 pM of each dNTP Diluted primer mix Primer A Variable 0 1 0 5 pM 0 2 pM is suitable for most PCR systems Primer B Variable 0 1 0 5 pM 0 2 pM is x suitable for most PCR systems Optional 5 pl 1x Coralload Concentrate 2 CoralLoad 2 Concentrate 10x i RNase free water Variable k Template DNA Template DNA Variable lt lpg reaction Total volume 50 pl N
33. ny Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and or its components For updated license terms see www qiagen com 2008 2010 QIAGEN all rights reserved ees EEE www qiagen com Australia Orders 1 800 243 800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 86 21 3865 3865 Fax 86 21 3865 3965 Technical 800 988 0325 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Orders 0800 914416 Fax 0800 914415 Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 60 920 930 Offers 01 60 920 928 Germany s Orders 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 800 789 544 Fax 02 334304 826 Technical 800 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 080 000 7146 Fax 02 2626 5703 T
34. ote If smaller or larger reaction volumes are used please adjust the amount of each component accordingly Contains 1 5 mM MgCl 5 When using a thermal cycler with a heated lid do not use mineral oil Proceed directly to step 6 Otherwise overlay with approximately 50 pl mineral oil 6 Program the thermal cycler according to the manufacturer s instructions The PCR cycling program outlined below has been pre optimized for most primer template systems However if an optimization of the annealing temperature is required please refer to Appendix B Page 31 24 TopTag PCR Handbook 06 2010 Table 8 Pre optimized cycling protocol Additional comments Initial denaturation 3 min 94 C 3 step cycling Denaturation 30 s 9A C Annealing 30 s 60 C The annealing temperature of 60 C is suitable for most PCR systems with Toplaa Master Mix If optimization of annealing temperature is required see Table 12 page 32 Appendix B for more information a Extension 1 min 72e For PCR products longer than 1 kb 3 use an extension time of R approximately 1 min per kb DNA z Number of cycles 25 35 See Appendix C page 35 8 Final extension 10min 72 C 7 Place the PCR tubes in the thermal cycler and start the cycling program Note After amplification samples can be stored overnight at 2 8 C or at 20 C for longer storage 8 When using Coralload Concentrate the PCR reaction can be directly loaded onto an ag
35. ou can find view and print the MSDS for each QIAGEN kit and kit component 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 Product Specifications Enzyme Toplaa DNA Polymerase is a recombinant thermostable 94 kDa DNA polymerase Concentration 5 units pl Compatible substrate analogs dNTP ddNTP Extension rate 2 4 kb min at 72 C 5 3 exonuclease activity Yes Extra A addition Yes 3 5 exonuclease activity No Nuclease contamination No Protease contamination No RNase contamination No Self priming activity No Storage and dilution buffer 20 mM Tris Cl 100 mM KCI 1 mM DTT 0 1 mM EDTA 50 glycerol v v stabilizers pH 9 0 20 C Buffers and reagents Toplaa PCR Buffer 10x concentrated Contains Tris Cl KCl NH SO 15 mM MgCl stabilizers pH 8 7 20 C Coralload Concentrate 10x concentrated Contains gel loading reagent orange dye red dye 6 TopTag PCR Handbook 06 2010 QSolution MgCl Solution TopTaa Master Mix Quality Control Enzyme Amplification efficiency assay PCR reproducibility assay Exonuclease activity assay Endonuclease activity assay RNase activity assay Protease activity assay Sel priming activity assay TopTaq PCR Handbook 06 2010 5x concentrated 25 mM 2x concentrated Contains TopTaq DNA Polym
36. oubleshooting Guide 26 Appendix A Starting Template 30 Appendix B Primer Design Concentration and Storage 31 Appendix C Number of PCR Cycles 35 Appendix D RT PCR 35 Appendix E Touchdown PCR 37 Appendix F Purification of PCR Products 37 Appendix G Control of Contamination 38 Ordering Information 39 TopTaq PCR Handbook 06 2010 3 Kit Contents TopTaq DNA Polymerase 250 U 1000 U 5000 U Catalog no 200203 200205 200207 TopTaq DNA Polymerase 250 units 1000 units 5000 units TopTaq PCR Buffer 10x 1 2 ml Ax12ml 1x22ml Coralload Concentrate 10x 1 2 ml Ax12ml 4x55 ml Q Solution 5x 2 ml 4x2 ml 1 x 40 ml MgCl 25mM 1 2 ml Ax 1 2 ml 1x 22 ml Handbook 1 1 1 Contains 15 mM MgCl TopTaq Master Mix Kit Catalog no 200403 Number of units 250 Number of 50 pl reactions 200 TopTaq Master Mid 2x 3 x 1 7 ml Coralload Concentrate 10x 1x 1 2 ml RNase Free Water 3x 1 9 ml Handbook 1 t Contains TopTaq DNA Polymerase TopTaq PCR Buffer with 3 mM MgCl and 400 pM each dNTP Shipping and Storage TopTaq DNA Polymerase and the TopTaq Master Mix Kit are shipped on dry ice but retain full activity at room temperature 15 25 C for at least 2 weeks They should be stored immediately upon receipt at 2 8 C When stored under these conditions and handled correctly these products can be kept at least until the expiration date see the inside of the kit lid without showing any reduction in performance TopTaq DNA
37. pet tips containing hydrophobic filters to minimize cross contamination Set up all reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier 28 TopTag PCR Handbook 06 2010 Comments and suggestions c Enzyme concentration too high Use 1 25 units TopTaq DNA Polymerase per 50 pl reaction When using TopTaq Master Mix always use 25 pl of TopTaa Master Mix per 50 pl reaction d Too many cycles Reduce the number of cycles in steps of 3 cycles e Mg concentration not optimal Perform PCR with different final concentra tions of Mg from 1 5 5 0 mM in 0 5 mM steps using the 25 mM MgCl solution provided f Primer concentration not optimal The primer concentration of 0 2 pM is or primers degraded suitable for most PCR systems with TopTaq DNA Polymerase However when primer concentration optimization is desired repeat the PCR with different primer concentrations from 0 1 0 5 pM of each primer in 0 1 pM steps In particular when performing highly sensitive PCR check for possible degradation of the primers on a denaturing polyacrylamide gel g Primer design not optimal Review primer design see Appendix B page 31 When working with chemicals
38. plate DNA should always be included TopTaq PCR Handbook 06 2010 19 3 a 8 a 2 g OZ n P v ET EL d oS Jo d n ts Table 4 Recommended reaction composition using TopTaq DNA Polymerase and Q Solution Component Volume reaction Final concentration Master mix o 10x TopTaq PCR Buffer 5 pl 1x 8 Optional 10x Coralload 5 pl 1x S Concentrate RE EE 5x Q Solution 10 pl Ix a lt 3 dNTP mix 10 mM of each 1 pl 200 pM of each dNTP 2 g Primer A Variable 0 1 0 5 pM 0 2 pM is 75 suitable for most PCR rd systems Primer B Variable 0 1 0 5 pM 0 2 pM is suitable for most PCR systems TopTaq DNA Polymerase 0 25 pl 1 25 units reaction RNase free water Variable Template DNA Template DNA added at Variable s1 pg reaction step 4 Total volume 50 pl Note If smaller reaction volumes are used please reduce the amount of each component accordingly Contains 15 mM MgCl TopTaq should only be used in combination with 10x TopTaq PCR buffer 3 Mix the master mix thoroughly and dispense appropriate volumes into PCR tubes Mix gently e g by pipetting the master mix up and down a few times It is not necessary to keep PCR tubes on ice as the unique PCR buffer formulation prevents nonspecific DNA synthesis at room temperature 4 Add template DNA lt 1 pg reaction to the individual tubes containing the master mix For RT PCR add an aliquot from the reverse transcriptase reaction
39. required and the risk of contamination and pipetting variability is minimized making PCR setup quick and easy TopTaq DNA Polymerase TopTaq DNA Polymerase is a recombinant 94 kDa thermostable DNA Polymerase TopTaq DNA Polymerase provides high PCR product yield and increased specificity without the need for optimization Toplaa Stabilizer binds to TopTaq DNA Polymerase at 4 C and room temperature preventing polymerase denaturation during long term storage Template DNA and primers are also prevented from binding to the polymerase at low temperature During the initial denaturation step the TopTaq Stabilizer dissociates from the polymerase without compromising polymerase activity TopTaq Master Mix TopTaq Master Mix consists of a ready to use premixed solution containing TopTaq DNA Polymerase dNTPs and the innovative TopTaq PCR Buffer Providing all components in a ready to use master mix reduces pipetting steps therefore reducing the risk of contamination High yields of PCR product are achieved even after storing the TopTaa Master Mix for 4 months at 25 C 4 C or 20 C TopTaq PCR Handbook 06 2010 9 CoralLoad Concentrate TopTaq DNA Polymerase and TopTaq Master Mix Kit are supplied with Coralload Concentrate which contains a gel loading reagent and two gel tracking dyes that facilitate estimation of DNA migration distance and optimization of agarose gel run time When using Coralload Concentrate the PCR products can be direc
40. rved Case A QSolution enables amplification of a reaction which previously failed z 2 2 e R ic 3 Le Case B Q Solution increases PCR specificity in certain primer template systems Case C G Solution has no effect on PCR performance N 9 Q E e A lt 4 a 8 8 Case D Q Solution causes reduced efficiency or failure of a previously successful amplification reaction In this case addition of Q Solution disturbs the previously optimal primer template annealing Therefore when using Q Solution for the first time for a particular primer template system always perform reactions in parallel with and without Q Solution 1 5 kb without Q Solution with Q Solution M markers 18 TopTag PCR Handbook 06 2010 Important points before starting When using Q Solution for the first time in a particular primer template system it is important to perform parallel amplification reactions with and without Q Solution The protocol offers convenient pre optimized primer concentrations and an annealing temperature that works for most primer template systems It is recommended to start with these pre optimized values It is essential that the provided 10x TopTaq PCR Buffer is used to ensure optimal PCR performance Set up all reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis Use disposable tips containing hydrophobic filters to
41. s and the relatively low efficiency of the reverse transcription reaction must be considered when calculating the appropriate amount of starting template for subsequent PCR The volume of the RT reaction transferred should not exceed 10 of the total PCR volume General guidelines are presented in Table 16 page 36 TopTaq PCR Handbook 06 2010 35 Table 16 General guidelines for performing RT PCR RNA purification QIAGEN offers the RNeasy system for total RNA isolation and reverse Oligotex Kits for messenger RNA isolation and transcription Omniscript Reverse Transcriptase for reverse transcription Follow the detailed protocol in the Omniscript Reverse Transcriptase Handbook When using an enzyme from another supplier follow the manufacturer s instructions The following guidelines may be helpful M Mix the following reagents in a microcentrifuge tube 4 0 pl 5x RT buffer 1 0 pl RNase inhibitor 5 units pl 2 0 pl DTT 0 1 M 1 0 pl each dNTP 10 mM 1 pg RNA 2 5 pl primer 0 2 pg pl Reverse transcriptase Add RNase free water to a final volume of 20 pl E Incubate following the manufacturer s instructions M Heat the reaction mix to 95 C for 5 min to inactivate the reverse transcriptase PCR E Prepare a PCR mixture following steps 1 3 in protocols M Add 2 5 pl from the RT reaction to each PCR tube containing the master mix M Continue with step 5 in the PCR protocols Oligotex resin is not available in
42. tly loaded onto an agarose gel without prior addition of loading buffer Coralload dyes do not interfere with most downstream enzymatic applications However for reproducible results purification of PCR products prior to enzymatic manipulation is recommended PCR Buffer The innovative TopTaq PCR Buffer facilitates the amplification of specific PCR products During the annealing step of every PCR cycle the buffer greatly increases the ratio of specific primer binding over nonspecific primer binding Owing to a uniquely balanced combination of KCI and NH SO the PCR buffer provides stringent primer annealing conditions over a wider range of temperatures and Mg concentrations than conventional PCR buffers The need to optimize PCR by varying the annealing temperature or the Mg concentration is dramatically reduced or offen not required Q Solution TopTaq DNA Polymerase is supplied together with Q Solution an innovative PCR additive that facilitates amplification of difficult templates by modifying the melting behavior of DNA This unique reagent will often enable or improve a suboptimal PCR caused by templates that have a high degree of secondary structure or that are GC rich Unlike other commonly used PCR additives such as DMSO Q Solution is used at just one working concentration it is nontoxic and PCR purity is guaranteed For further information please read the protocol PCR Using TopTaq DNA Polymerase and QSolution pa
43. uced from an amino acid sequence To enable such templates to be amplified by PCR degenerate primers can be used These are actually mixtures of several primers whose sequences differ at the position that correspond fo the uncertainties in the template sequence PCR using TopTaa Master Mix often improves the specificity of PCR amplifications that employ degenerate primers by reducing the formation of nonspecific PCR products and primer dimers Table 13 gives recommendations for further optimizing PCR using degenerate primers Table 14 shows the codon redundancy of each amino acid TopTaq PCR Handbook 06 2010 33 Table 13 Guidelines for design and use of degenerate primers Seguence E Avoid degeneracy in the 3 nucleotides at the 3 end M If possible use Met or Trp encoding triplets at the 3 end M To increase primer template binding efficiency reduce degeneracy by allowing mismatches between the primer and template especially towards the 5 end but not at the 3 end M Try to design primers with less than 4 fold degeneracy at any given position Concentration M Begin PCR with a primer concentration of 0 2 pM M In case of poor PCR efficiency increase primer concentrations in increments of 0 25 pM until satisfactory results are obtained Table 14 Codon redundancy Amino acid Number of codons Met Trp Cys Asp Glu Phe His Lys Asn Gln Tyr 2 lle 3 Ala Gly Pro Thr Val 4 Leu Arg Ser 6

TopTaq PCR Handbook

Contents

Download Pdf Manuals

Related Search

Related Contents