Introduction

 PCR (Polymerase Chain Reaction) is a powerful method for the amplification of nucleic acids.  It is often used to clone DNA, facilitate DNA sequencing, and generated labeled probes.  This wide variety of applications requires an occasional adjustment to the PCR process.  Optimizing PCR for your specific application can mean the difference between success and failure.  To ensure your success, a selection of PCR enzymes and optimization kits is available.  In this unit, we highlight protocols for high fidelity.
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Protocol: Generating PCR Products with Pfx™50 DNA Polymerase

Pfx50™ DNA Polymerase is a fusion enzyme consisting of recombinant DNA polymerase from the archaean Thermococcus zilligii fused to an accessory protein. The highly thermostable polymerase possesses a proofreading 3’ → 5’ exonuclease activity, while the accessory protein stabilizes primer-template complexes in PCR.
 
Pfx50™ DNA Polymerase offers 50 times better fidelity than Taq DNA polymerase,coupled with high specificity and an extremely fast elongation rate (as fast as 15 seconds per kb). In addition, the fusion enzyme has an intrinsic hot-start capability for room-temperature reaction assembly.

Note:

  • 10X Pfx50™ PCR buffer contains BSA; store at –20ºC.
  • Pfx50™ DNA Polymerase produces blunt-end PCR products, which can be used with Directional TOPO® Cloning and Zero Blunt® TOPO®Cloning technologies.

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  Amount  
Component 100 Rxn Kit 500 Rxn Kit
Pfx50™ DNA Polymerase (5 U/µl)100 µl500 µl
10X Pfx50™ PCR Mix  1.3 ml  2 × 1.3 ml
50-mM Magnesium Sulfate 500 µl2 × 1.25 ml
Anti-sense primer (10 µM)1 ml1 ml


Pfx50™ DNA Polymerase Storage Buffer
 
20 mM Tris-HCl (pH 8.0), 40 mM KCl, 0.1 mM EDTA, 1 mM DTT, stabilizers, and 50% (v/v) glycerol
 
Unit Definition

One unit of Pfx50™ DNA Polymerase incorporates 10 nmol of deoxyribonucleotide into acid-insoluble material in 30 min at 74°C.
 
General Recommendations and Guidelines for PCR

General PCR parameters and troubleshooting information are documented in Innis, et al.
 
Template: 
Pfx50™ DNA Polymerase is suitable for amplifying targets up to 4 kb from the following templates:

Template                  Amount
Genomic DNA          1–200 ng
Plasmid DNA            1–100 pg
cDNA                       3­–5 µl from 10 ng to 1 µg starting total RNA

Amplification of longer targets (up to 7 kb) may be possible, but may require more template and longer elongation times.
 
Primers: 
Use 0.3 µM per primer as a general starting point. For larger amounts of template (e.g., 200 ng genomic DNA), increasing the concentration up to 0.5 µM per primer may improve yield.
 
Annealing Temperature: 
The annealing temperature is slightly higher than with typical PCR. The optimal annealing temperature should be ~2ºC lower than the Tm of the primers used. A range of 60–68ºC is recommended.
 
MgSO4
MgSO4 is included in the 10X Pfx50™ PCR Mix at a final concentration of 1.2 mM, which is sufficient for most templates. For further optimization, add 0.1 µl to 1.0 µl of 50-mM MgSO4.
 
Extension Time: 
As little as 15 seconds per kb may be used; 30 seconds per kb is suitable for most targets. Use up to 60 seconds per kb for maximum yield.
 
The following procedure is suggested as a starting point when using Pfx50™ DNA Polymerase in any PCR amplification.
 

  1. Program the thermal cycler as follows (see the note on annealing temperature above): 
    Initial denaturation:                     94ºC for 2 minutes
    35 cycles of:
    Denaturation:                              94ºC for 15 seconds
    Annealing:                                   60–68ºC (Tm of primers minus 2ºC) for 10–30 seconds
    Extension:                                    68ºC for 30–60 seconds per kb of PCR product
    Final extension:                           68ºC for 5 minutes
     
     
  2. Add the following components to an autoclaved microcentrifuge tube at room temperature (for multiple reactions, prepare a Master Mix of common components to enable accurate pipetting):

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    Component
    VolumeFinal Conc.
    10X Pfx50™ PCR Mix5 µl1X
    10 mM dNTP Mix1.5 µl0.3 mM each
    Primer mix (10 µM each)1.5 µl0.3 µM each
    Template DNA ≥1 µlAs required
    Pfx50™ DNA Polymerase (5 U/µl)1 µl5 units
    Autoclaved distilled water50  µl

     
  3. Cap the tube, tap gently to mix, and centrifuge briefly to collect the contents.
  4. Place the tube in the thermal cycler and run the program from Step 1. After cycling, maintain the reaction at 4ºC. Samples can be stored at  –20ºC until use.


Analyze products using E-Gel® Pre-Cast agarose gels or standard agarose gel electrophoresis. Visualize by staining with SYBR Safe™ DNA gel stain or ethidium bromide.
 

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Reference

  1. Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. S. (eds) (1990) PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, CA
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