Platinum® Pfx DNA Polymerase
Cat. No. 11708-013
Size: 100 reactions 11708-021
250 reactions11708-039
500 reactions
Conc: 2.5 U/μl
Store at -20°C
Note: Read and follow reaction conditions carefully to ensureoptimal performance.
Description Platinum® Pfx DNA Polymerase is a proprietary enzyme preparation containing recombinant DNA polymerase from Thermococcus sp. strainKOD (1,2). Platinum® Pfx DNA Polymerase possesses proofreading3′ to 5′ exonuclease activity and provides higher fidelity than Pfu DNApolymerase (3). It is a highly processive enzyme with fast chainextension capability.Platinum® Pfx DNA Polymerase is provided in inactive form, due tospecific binding of the Platinum® antibody. Polymerase activity isrestored after a PCR denaturation step at 94°C, providing an automatic.hot start. and increasing specificity, sensitivity, and yield (4). Thehigh accuracy, specificity, and yield of Platinum® Pfx DNA Polymerasemake it ideal for demanding PCR applications such as site-directedmutagenesis and PCR expression cloning.For problematic and/or GC-rich templates, PCRx Enhancer Solution isincluded with each kit (see the guidelines for use on page 2). The number of reactions per kit is based on a standard reaction size of 50 μl.Kit SizeComponents 100 Rxns 250 Rxns 500 RxnsPlatinum Pfx DNA Polymerase 100 units 250 units 500 units50 mM Magnesium Sulfate 1 ml 1 ml 1 ml10X Pfx Amplification Buffer 1 ml 2 × 1 ml 3 × 1 ml10X PCRx Enhancer Solution 1 ml 2 × 1 ml 3 × 1 mlPart no. 11708.pps Rev. date 07/11/03T his product is distributed for laboratory research only.
CAUTION: Not for diagnostic use. The safety and efficacy of this product in diagnostic orother clinical uses has not been established.For technical questions about this product, call the Invitrogen Tech-LineSM U.S.A. 800 955 6288Page 2 of 4Platinum® Pfx DNA Polymerase Storage Buffer50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA,stabilizers, and 50% (v/v) glycerol.Unit DefinitionOne unit of Platinum Pfx DNA Polymerase incorporates 10 nmol ofdeoxyribonucleotide into acid-precipitable material in 30 min at 74°C.Quality ControlThe DNA polymerase:antibody complex is evaluated in a DNApolymerization activity assay that measures the percent of DNApolymerase inhibition versus an uninhibited control. Platinum® PfxDNA Polymerase is functionally tested in an amplification using 100 ngof K562 genomic DNA as a template.Guidelines for Using PCRx Enhancer SolutionFor problematic and/or GC-rich templates, PCRx Enhancer Solutionprovides higher primer specificity, a broader range of optimalmagnesium concentrations, broad annealing temperatures, andimproved thermostability.Use of PCRx Enhancer Solution is optional; use in combination with10X Pfx Amplification Buffer, not as a substitute. PCRx EnhancerSolution lowers the DNA melting temperature (Tm), reducing themaximum primer annealing temperature approximately 2°C per 1XPCRx Enhancer Solution concentration, while at the same timeexpanding the effective annealing temperature over a much broaderrange. To determine the optimal reaction concentrations andconditions, we recommend starting with an annealing temperature of55°C to 60°C and varying the amount of 10X PCRx EnhancerSolution. For targets with higher GC content (60 to 90%), werecommend testing 10X PCRx Enhancer Solution at final concentrationsof 0.5X, 1X, 2X, and 3X.Page 3 of 4PCR ProtocolThe following procedure is suggested as a guideline and starting pointwhen using Platinum Pfx DNA Polymerase in any PCR amplification.1. Add the following components to an autoclaved microcentrifuge tubeeither at ambient temperature or on ice:Component Volume Final Concentration10X Pfx Amplification Buffer 5 μl 1X10 mM dNTP mixture* 1.5 μl 0.3 mM each50 mM MgSO4 1 μl 1 mMPrimer mix (10 μM each)* 1.5 μl 0.3 μM eachTemplate DNA (10 pg - 200 ng) ≥1 μl As requiredPlatinum® Pfx DNA Polymerase** 0.4.1 μl 1.0.2.5 unitsAutoclaved, distilled water to 50 μl*Platinum® Pfx DNA Polymerase will not function in reactionsthat contain dUTP in either the dNTP mix or the primers.**For most targets 1 unit is sufficient. When amplifying targetsabove 3 kb, more enzyme may be required.2. Mix tube contents and overlay with mineral or silicone oil, if necessary.3. Cap the tube and centrifuge briefly to collect the contents.4. Denature the template for 2 min at 94°C. Perform 25.35 cycles of PCRamplification as follows:Three-step cycling Two-step cyclingDenature: 94°C for 15 s Denature: 94°C for 15 sAnneal: 55°C for 30 s Extend: 68°C for 1 min per kbExtend: 68°C for 1 min per kbNote: Two-step cycling can be used for long primers with highTm.5. Maintain the reaction at 4°C after cycling. Samples can be stored at-20°C until use.6. Analyze the products by agarose gel electrophoresis.
Cat. No. 11708-013
Size: 100 reactions 11708-021
250 reactions11708-039
500 reactions
Conc: 2.5 U/μl
Store at -20°C
Note: Read and follow reaction conditions carefully to ensureoptimal performance.
Description Platinum® Pfx DNA Polymerase is a proprietary enzyme preparation containing recombinant DNA polymerase from Thermococcus sp. strainKOD (1,2). Platinum® Pfx DNA Polymerase possesses proofreading3′ to 5′ exonuclease activity and provides higher fidelity than Pfu DNApolymerase (3). It is a highly processive enzyme with fast chainextension capability.Platinum® Pfx DNA Polymerase is provided in inactive form, due tospecific binding of the Platinum® antibody. Polymerase activity isrestored after a PCR denaturation step at 94°C, providing an automatic.hot start. and increasing specificity, sensitivity, and yield (4). Thehigh accuracy, specificity, and yield of Platinum® Pfx DNA Polymerasemake it ideal for demanding PCR applications such as site-directedmutagenesis and PCR expression cloning.For problematic and/or GC-rich templates, PCRx Enhancer Solution isincluded with each kit (see the guidelines for use on page 2). The number of reactions per kit is based on a standard reaction size of 50 μl.Kit SizeComponents 100 Rxns 250 Rxns 500 RxnsPlatinum Pfx DNA Polymerase 100 units 250 units 500 units50 mM Magnesium Sulfate 1 ml 1 ml 1 ml10X Pfx Amplification Buffer 1 ml 2 × 1 ml 3 × 1 ml10X PCRx Enhancer Solution 1 ml 2 × 1 ml 3 × 1 mlPart no. 11708.pps Rev. date 07/11/03T his product is distributed for laboratory research only.
CAUTION: Not for diagnostic use. The safety and efficacy of this product in diagnostic orother clinical uses has not been established.For technical questions about this product, call the Invitrogen Tech-LineSM U.S.A. 800 955 6288Page 2 of 4Platinum® Pfx DNA Polymerase Storage Buffer50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA,stabilizers, and 50% (v/v) glycerol.Unit DefinitionOne unit of Platinum Pfx DNA Polymerase incorporates 10 nmol ofdeoxyribonucleotide into acid-precipitable material in 30 min at 74°C.Quality ControlThe DNA polymerase:antibody complex is evaluated in a DNApolymerization activity assay that measures the percent of DNApolymerase inhibition versus an uninhibited control. Platinum® PfxDNA Polymerase is functionally tested in an amplification using 100 ngof K562 genomic DNA as a template.Guidelines for Using PCRx Enhancer SolutionFor problematic and/or GC-rich templates, PCRx Enhancer Solutionprovides higher primer specificity, a broader range of optimalmagnesium concentrations, broad annealing temperatures, andimproved thermostability.Use of PCRx Enhancer Solution is optional; use in combination with10X Pfx Amplification Buffer, not as a substitute. PCRx EnhancerSolution lowers the DNA melting temperature (Tm), reducing themaximum primer annealing temperature approximately 2°C per 1XPCRx Enhancer Solution concentration, while at the same timeexpanding the effective annealing temperature over a much broaderrange. To determine the optimal reaction concentrations andconditions, we recommend starting with an annealing temperature of55°C to 60°C and varying the amount of 10X PCRx EnhancerSolution. For targets with higher GC content (60 to 90%), werecommend testing 10X PCRx Enhancer Solution at final concentrationsof 0.5X, 1X, 2X, and 3X.Page 3 of 4PCR ProtocolThe following procedure is suggested as a guideline and starting pointwhen using Platinum Pfx DNA Polymerase in any PCR amplification.1. Add the following components to an autoclaved microcentrifuge tubeeither at ambient temperature or on ice:Component Volume Final Concentration10X Pfx Amplification Buffer 5 μl 1X10 mM dNTP mixture* 1.5 μl 0.3 mM each50 mM MgSO4 1 μl 1 mMPrimer mix (10 μM each)* 1.5 μl 0.3 μM eachTemplate DNA (10 pg - 200 ng) ≥1 μl As requiredPlatinum® Pfx DNA Polymerase** 0.4.1 μl 1.0.2.5 unitsAutoclaved, distilled water to 50 μl*Platinum® Pfx DNA Polymerase will not function in reactionsthat contain dUTP in either the dNTP mix or the primers.**For most targets 1 unit is sufficient. When amplifying targetsabove 3 kb, more enzyme may be required.2. Mix tube contents and overlay with mineral or silicone oil, if necessary.3. Cap the tube and centrifuge briefly to collect the contents.4. Denature the template for 2 min at 94°C. Perform 25.35 cycles of PCRamplification as follows:Three-step cycling Two-step cyclingDenature: 94°C for 15 s Denature: 94°C for 15 sAnneal: 55°C for 30 s Extend: 68°C for 1 min per kbExtend: 68°C for 1 min per kbNote: Two-step cycling can be used for long primers with highTm.5. Maintain the reaction at 4°C after cycling. Samples can be stored at-20°C until use.6. Analyze the products by agarose gel electrophoresis.
Posts
No comments:
Post a Comment