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Manual: Pfu DNA Polymerase Pfu DNA Polymerase Instruction Manual Catalog #600135, #600136, and #600140 (Native Pfu DNA Polymerase) and #600153, #600154, #600159, and #600160 (Cloned Pfu DNA Polymerase) Revision C Research Use Only. Not for Use in Diagnostic Procedures. 600135-12 LIMITED PRODUCT WARRANTY This warranty limits our liability to replacement of this product. No other warranties of any kind, express or implied, including without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided by Agilent. Agilent shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or the inability to use this product. ORDERING INFORMATION AND TECHNICAL SERVICES United States and Canada Agilent Technologies Stratagene Products Division 11011 North Torrey Pines Road La Jolla, CA 92037 Telephone (858) 373-6300 Order Toll Free (800) 424-5444 Technical Services (800) 894-1304 email [email protected] World Wide Web www.genomics.agilent.com Europe Location Telephone Austria 01 25125 6800 Benelux 02 404 92 22 Denmark 45 70 13 00 30 Finland 010 802 220 France 0810 446 446 Germany 0800 603 1000 Italy 800 012575 Netherlands 020 547 2600 Spain 901 11 68 90 Sweden 08 506 4 8960 Switzerland 0848 8035 60 UK/Ireland 0845 712 5292 All Other Countries Please contact your local distributor. A complete list of distributors is available at www.genomics.agilent.com. Pfu DNA Polymerase CONTENTS Materials Provided.............................................................................................................................. 1 Storage Conditions.............................................................................................................................. 1 Notices to Purchaser ........................................................................................................................... 1 Introduction......................................................................................................................................... 3 Critical Optimization Parameters for Pfu DNA Polymerase-Based PCR ..................................... 3 Extension Time...................................................................................................................... 3 Enzyme Concentration .......................................................................................................... 3 Reaction Buffer ..................................................................................................................... 4 Primer–Template Purity and Concentration.......................................................................... 5 Primer Design........................................................................................................................ 5 Additional Optimization Parameters for Pfu DNA Polymerase-Based PCR ................................ 6 PCR Cycling Parameters ....................................................................................................... 6 Order of Addition of Reaction Mixture Components............................................................ 6 Deoxynucleoside Triphosphates............................................................................................ 6 Salt Concentrations................................................................................................................ 7 Adjuncts and Cosolvents ....................................................................................................... 7 Application Notes ................................................................................................................................ 9 Long PCR.............................................................................................................................. 9 Thermostability ..................................................................................................................... 9 Modified Nucleotide Incorporation....................................................................................... 9 Terminal Transferase Activity............................................................................................... 9 Reverse Transcriptase Activity.............................................................................................. 9 PCR Protocol Using Pfu DNA Polymerase..................................................................................... 10 Troubleshooting ................................................................................................................................ 12 Preparation of Media and Reagents................................................................................................ 13 References.......................................................................................................................................... 13 Endnotes............................................................................................................................................. 14 MSDS Information............................................................................................................................ 14 Pfu DNA Polymerase MATERIALS PROVIDED Native Pfu DNA Polymerase Quantity Materials provided Catalog #600135 Catalog #600136 Catalog #600140 Native Pfu DNA polymerase (2.5 U/μl) 100 Ua 500 Ub 1000 Uc Native Plus 10× Pfu buffer 1 ml 2 × 1 ml 4 × 1 ml a Sufficient native Pfu DNA polymerase is provided for up to 40 100-μl reactions. b Sufficient native Pfu DNA polymerase is provided for up to 200 100-μl reactions. c Sufficient native Pfu DNA polymerase is provided for up to 400 100-μl reactions. Cloned Pfu DNA Polymerase Quantity Catalog Catalog Catalog Catalog Materials provided #600153 #600154 #600159 #600160 Cloned Pfu DNA polymerase (2.5 U/μl) 100 Ua 500 Ub 1000 Uc 5000 Ud 10× Cloned Pfu buffere 1 ml 2 × 1 ml 4 × 1 ml 20 × 1 ml a Sufficient cloned Pfu DNA polymerase is provided for up to 40 100-μl reactions. b Sufficient cloned Pfu DNA polymerase is provided for up to 200 100-μl reactions. c Sufficient cloned Pfu DNA polymerase is provided for up to 400 100-μl reactions. d Sufficient cloned Pfu DNA polymerase is provided for up to 2000 100-μl reactions. e See Preparation of Media and Reagents. STORAGE CONDITIONS All components: –20°C NOTICES TO PURCHASER Limited Label License for Native and Cloned Pfu DNA Polymerase Products This product is covered by the claims of one or more of the following U.S. Patents: 5,545,552; 5,866,395; 5,948,663; 6,489,150; 6,183,997; 6,333,165; 6,379,553; 6,444,428. Purchase of this product conveys to the purchaser only the non-transferable right under these patents to use the product for research use only by the purchaser. No rights are granted to the purchaser hereunder to sell, modify for resale or otherwise transfer this product, either alone or as a component of another product, to any third party. Agilent reserves all other rights, and this product may not be used in any manner other than as provided herein. For information on obtaining a license to use this product for purposes other than research, please contact Stratagene Products Division Business Development, 11011 North Torrey Pines Road, La Jolla, California 92037. Phone (858) 535-5400. Revision C © Agilent Technologies, Inc. 2010. Pfu DNA Polymerase 1 Notice to Purchaser: Limited License Purchase of this product includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchaser’s own internal research. No other patent rights (such as 5’ Nuclease Process patent rights) are conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. 2 Pfu DNA Polymerase INTRODUCTION Pfu DNA polymerase,* a proofreading DNA polymerase isolated from Pyrococcus furiosus, is an ideal choice for a variety of techniques requiring high-fidelity DNA synthesis by the polymerase chain reaction (PCR).1–3 These applications include cloning, gene expression, and site-directed mutagenesis. Successful PCR using Pfu DNA polymerase is readily performed requiring only slight modifications from PCR protocols optimized with Taq DNA polymerase. Various PCR parameters that are important in increasing the yield and specificity of Pfu DNA polymerase- based PCR amplification reactions are described in this instruction manual. CRITICAL OPTIMIZATION PARAMETERS FOR Pfu DNA POLYMERASE- BASED PCR All PCR amplification reactions, whether performed using Taq or Pfu DNA polymerase, require optimization to achieve the highest product yield and specificity. Critical optimization parameters for successful PCR using Pfu DNA polymerase are outlined in the following sections and include the use of an extension time that is adequate for full-length DNA synthesis, sufficient enzyme concentration, optimization of the reaction buffer, adequate primer–template purity and concentration, and optimal primer design. Extension Time Extension time is the most critical parameter affecting the yield of PCR product obtained using Pfu DNA polymerase. For Taq DNA polymerase- based PCR amplifications, an extension time of 0.5-1.0 minute/kb of template amplified is usually sufficient for maximum synthesis of a PCR target. In contrast, Pfu DNA polymerase-based PCR amplifications require a minimum extension time of 1-2 minutes/kb of amplified template to
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