Transcription of Platinum Taq DNA Polymerase High Fidelity
1 Protocol Pub. No. MAN0000948 Rev. Platinum Taq DNA Polymerase Print Options high Fidelity Catalog Number Size Enzyme Characteristics Package 11304-011 100 rxns Kit Contents Contents 11304-029 500 rxns Hot-start: Antibody 11304-102 5,000 rxns Length: Up to 20 kb Storage Conditions Store all contents at -20 C. Fidelity vs. Taq: 6X. Format: Separate components Template: cDNA, gDNA, DNA PCR Reaction Setup Forward and reverse gene-specific primers 10 mM dNTP mix (Cat. no. 18427-088) Use the measurements below to prepare your PCR experiment, or enter Required Autoclaved, distilled water your own parameters in the column provided.
2 Materials E-Gel General Purpose Gels, (Cat. no. G5018-01).. TrackIt 1 Kb Plus DNA Ladder (Cat. no. 10488-085) Component 25- L rxn 50- L rxn Custom Final Conc. or nuclease-free microcentrifuge tubes Autoclaved, distilled to 25 L to 50 L to L . water Timing Varies depending on amplicon length 10X high Fidelity L 5 L L 1X. Selection PCR Enzymes and Master Mixes PCR Buffer Guide Go online to view related products. 50 mM MgSO4 1 L 2 L L mM. Platinum Taq DNA Polymerase high Fidelity contains mM. recombinant Taq DNA Polymerase , Pyrococcus species GB-D 10 mM dNTP Mix L 1 L L.
3 Polymerase , and Platinum Taq Antibody. each This enzyme allows amplification of simple and complex 10 M forward primer L 1 L L M. Product DNA templates over a large range of target sizes and Description provides 6X higher Fidelity over Taq. 10 M reverse primer L 1 L L M. Activity is restored after the denaturation step in PCR Template DNA varies varies < 500 ng cycling at 94 C, providing an automatic hot start and offering increased sensitivity, specificity, and yield, while Platinum Taq DNA. allowing assembly of reactions at room temperature.
4 Polymerase high L L L 1 U/rxn Select the correct Polymerase , PCR instrument, and cycling Fidelity (5 U/ L). conditions for your application. PCR Protocol Take precautions to avoid cross-contamination by using See page 2 to view a procedure for preparing and running your PCR. aerosol-resistant barrier tips and analyzing PCR products Important in a separate area from PCR assembly. experiment. Guidelines For gDNA or cDNA, use a primer concentration of M. For Plasmid or DNA, increase to M. Optimization Strategies Do not perform the initial denaturation for more than 30 Refer to the pop-up for guidelines to seconds if the target is greater than 12 kb.
5 Optimize your PCR reactions. Visit our product page for additional Online information and protocols. For support, Limited Warranty, Disclaimer, Resources visit and Licensing Information For Research Use Only. Not for use in diagnostic procedures. Platinum Taq DNA Polymerase high Fidelity Protocol 2014. Platinum Taq DNA Polymerase high Fidelity Protocol The example PCR procedure below shows appropriate volumes for a single 50- L reaction. For multiple reactions, prepare a master mix of components common to all reactions to minimize pipetting error, and then dispense appropriate volumes into each mL PCR reaction tube prior to adding template DNA and primers.
6 Timeline Steps Procedure Details 1 Thaw reagents Thaw, mix, and briefly centrifuge each component before use Add the following components to each PCR reaction tube. Note: Consider the volumes for all components listed in steps 2 and 3 to determine the correct amount of water required to reach your final reaction volume. Component 50- L rxn Final Concentration Autoclaved, distilled water to 50 L. 10X high Fidelity PCR Buffer 5 L 1X. 2 Prepare PCR master mix 10 mM dNTP mix 1 L mM each 50 mM MgSO4 2 L 2 mM. Platinum Taq DNA high Fidelity Polymerase (5 U/ L) L 1 U*.
7 * 1 U is sufficient for amplifying most targets. In some cases, more enzyme may be required (up to units). Use 1 U of enzyme for targets > 12 kb. Mix and briefly centrifuge the components. Add your template DNA and primers to each tube for a final reaction volume of 50 L. Component 50- L rxn Final Concentration M (Genomic and cDNA) . 10 M forward primer 1 2 L. Add template DNA and M (Plasmid and DNA). 3. primers 10 M reverse primer 1 2 L. M (Genomic and cDNA) . M (Plasmid and DNA). Template DNA varies < 500 ng Cap each tube, mix, and then briefly centrifuge the contents.
8 Step Temperature ( C) Time Initial Denaturation 94 30 seconds 2 minutes*. Denature 94 15 seconds 25 35 ~55 (depending Incubate reactions in a PCR Anneal 30 seconds 4 on primer Tm). thermal cycler Cycles Extend 68 1 minute/kb Hold 4 indefinitely * For targets > 12 kb, do not exceed 30 seconds for initial denaturation. Analyze with gel Analyze 10 L using agarose gel electrophoresis. 5. electrophoresis Use your PCR reaction immediately for down-stream applications, or store it at -20 C. For support, visit 28 February 2014 -2.
