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 specificity.
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Ordering Information

Sku Name Size Price Qty
12339024 AccuPrime™ Taq DNA Polymerase System 1,000 reactions USD 1,166.00
12339016 AccuPrime™ Taq DNA Polymerase System 200 reactions USD 248.00

Protocol with AccuPrime™ Taq DNA Polymerase System

The AccuPrime™ Taq  DNA Polymerase System provides qualified reagents for the amplification of nucleic acid templates by polymerase chain reaction (PCR). AccuPrime™ Taq  DNA polymerase contains anti-Taq  DNA polymerase antibodies. The 10X AccuPrime™ buffers contain thermostable AccuPrime™ protein, Mg++, and deoxyribonucleotide triphosphates at concentrations sufficient to allow amplification during PCR. Two individual buffer systems (10X AccuPrime™ PCR Buffer I and II) are provided for amplification of specific types of templates.* Reagents sufficient for 200 or 1,000 amplification reactions of 25 µl each are provided.
 
Anti-Taq  DNA polymerase antibodies inhibit polymerase activity, providing an automatic “hot start” (1,2) and permitting room temperature set-up. The thermostable AccuPrime™ protein enhances specific primer-template hybridization during every cycle of PCR. Antibody/AccuPrime™ protein-mediated amplification dramatically improves PCR specificity, improves the fidelity of Taq by two fold, and provides the most robust PCR for multiplex PCR and suboptimal primer sets

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  Amount  
Component 200 rxn kit 1,000 rxn kit
AccuPrime™ Taq DNA Polymerase100 µl500 µl
10X AccuPrime™ PCR Buffer I*500 µl2 × 1.25 ml
10X AccuPrime™ PCR Buffer II*500 µl2 × 1.25 ml
Anti-sense primer (10 µM)1 µl1 µl
50 mM Magnesium Chloride500 µl500 µl 


*10X AccuPrime™ PCR Buffer I is designed for small genomic DNA amplicons (<200 bp), cDNA, or plasmids.
 
 10X AccuPrime™ PCR Buffer II is designed for genomic DNA (200 bp–4 kb).
 
Storage Buffer

20 mM Tris­-HCl (pH 8.0), 0.1 mM EDTA, 1 mM DTT, stabilizers, 50% (v/v) glycerol
10X AccuPrime™ PCR Buffer I and II
200 mM Tris-HCl (pH 8.4), 500 mM KCl, 15 mM MgCl2, 2 mM dGTP, 2 mM dATP,        2 mM dTTP, 2 mM dCTP, thermostable AccuPrime™ protein, 10% glycerol
 
Quality ControlAccuPrime™ Taq DNA Polymerase is evaluated in a PCR functional assay. AccuPrime™ Taq DNA Polymerase and 10X AccuPrime™ PCR Buffers are functionally tested for amplification. AccuPrime™ Taq DNA Polymerase and AccuPrime™ protein are tested for the absence of double- and single-stranded endonuclease activity as well as the absence of contaminating 5´- and 3´-exonuclease activity.

PCR Precautions


Since PCR is a powerful technique capable of amplifying trace amounts of DNA, all appropriate precautions should be taken to avoid cross-contamination. Ideally, amplification reactions should be assembled in a DNA-free environment. Use of aerosol-resistant barrier tips is recommended. Take care to avoid contamination with the primers or template DNA used in individual reactions. PCR products should be analyzed in an area separate from the reaction assembly area.
 
Protocol
 
The following procedure is suggested as a general protocol when using AccuPrime™ Taq DNA Polymerase in any PCR amplification. Optimal reaction conditions (incubation times and temperatures; the amounts of AccuPrime™ Taq DNA Polymerase, primers, MgCl2, and template DNA) will vary and may need to be optimized. Reaction sizes may be altered to suit user preference, as shown in the tables below.
 

  1. Add the following components to a sterile thin walled 0.25-ml or 0.5-ml PCR tube either at room temperature or on ice: 
    For Small Genomic DNA ( < 200 bp), Plasmids, or cDNA):
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    Component
    10-µl Reaction
    25-µl Reaction
    50-µl Reaction
    10X AccuPrime PCR Buffer I
    1 µl
    2.5 µl
    5 µl
    Primer Mix (10 µM each)
    0.2 µl
    0.5 µl
    1 µl
    Template DNA
    10 pg–200 ng
    10 pg–200 ng
    10 pg–200 ng
    AccuPrime Taq DNA Polymerase
    0.25 µl
    0.5 µl
    1 µl
    Autoclaved distilled water
    To 10 µl
    To 25 µl
    To 50 µl

    For Genomic DNA (200 bp-4 kb):

    Component
    10-µl Reaction
    25-µl Reaction
    50-µl Reaction
    10X AccuPrime PCR Buffer II
    1 µl
    2.5 µl
    5 µl
    Primer Mix (10 µM each)
    0.2 µl
    0.5 µl
    1 µl
    Template DNA
    1–200 ng
    1–200 ng
    1–200 ng
    AccuPrime Taq DNA Polymerase
    0.25 µl
    0.5 µl
    1 µl
    Autoclaved distilled water
    To 10 µl
    To 25 µl
    To 50 µl

    If desired, a master mix can be prepared for multiple reactions to minimize reagent loss and enable accurate pipetting.
     
  2. Mix contents of the tubes and overlay with 50 µl of mineral or silicone oil, if necessary.
  3. Cap the tubes and centrifuge briefly to collect the contents.
  4. Incubate tubes in a thermal cycler at 94°C for 2 min to completely denature the template and activate the enzyme.
  5. Perform 25-35 cycles of PCR amplification as follows:
     
          Denature:    94°C for 15-30 s
          Anneal:       55°C-60°C for 15-30 s
          Extend:        68°C for 1 min per kb<
  6. Maintain the reaction at 4°C after cycling. The samples can be stored at -20°C until use.
  7. Analyze the amplification products by agarose gel electrophoresis and visualize by ethidium bromide staining. Use appropriate molecular weight standards.


 
Multiplex PCR Protocol

The following specialized procedure is suggested as a guideline and as a starting point when using AccuPrime™ Taq DNA Polymerase in multiplex PCR amplification. Optimal reaction conditions (incubation times and temperatures; the amounts of AccuPrime™ Taq DNA Polymerase, primers, MgCl2, and template DNA) will vary and may need to be optimized. Reaction sizes may be altered to suit user preference, as shown in the tables below.
 

  1. Add the following components to a sterile, thin-walled 0.25-ml or 0.5-ml PCR tube either at room temperature or on ice:
    Component
    Amount
    10X AccuPrime PCR Buffer I (for genomic DNA <200 bp, cDNA, or plasmids) or
    10X AccuPrime PCR Buffer II (for genomic DNA 200 bp–4 kb)
    5 µl
    Primer mix (10 µM each)
    1 µl each (0.2 µM each)
    Template DNA
    100-200 ng
    AccuPrime Taq  DNA Polymerase*
    1-2.5 µl
    Autoclaved, distilled water
    to 50 µl


    *For primer mixes up to 5 sets, 1 µl of enzyme is sufficient.
     
    If desired, a master mix can be prepared for multiple reactions, to minimize reagent loss and to enable accurate pipetting.
  2. Continue with steps 2-7 of the General Protocol.
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References

  1.     Chou, Q., Russel, M., Birch, D., Raymond, J., Bloch, W. (1992) Nucl. Acids Res. 20, 1717.
 
  2.     Sharkey, D.J., Scalice, E.R., Christy, K.G., Atwood, S.M., Daiss, J.L. (1994) BioTechnology 12, 506.
 
  3.     Westfall, B., Sitaraman, K., Solus, J., Hughes, J., Rashtchian, A. (1997) Focus® 19, 46.
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