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 yield depending upon your needs:  

  1.    Opt for Platinum® Taq DNA Polymerase for high yield.

  2.    Use Platinum® Taq DNA Polymerase High Fidelity for high yield and fidelity.

  3.    Select AccuPrime™  Taq High Fidelity for high yield, specificity, and fidelity.
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Protocol with Platinum® Taq DNA Polymerase

PCR Precautions

An extension temperature of 68°C, the use of thin-walled reaction tubes for targets above 5 kb, the use of one unit of enzyme for targets above 12 kb, and an increased primer concentration of 0.4 µM for plasmid and lambda targets are all recommended conditions.
 
Protocol

The following procedure is suggested as a guideline and starting point when using Platinum® Taq  DNA Polymerase High Fidelity in any PCR amplification. Reaction size may be altered to suit user preferences.
 
  1. Add the following components to a autoclaved microcentrifuge tube at either ambient temperature, or on ice:

  2. Components Volume Final Concentration
    10X High Fidelity PCR Buffer5 µl1X
    10 mM dNTP mixture 1 µl 0.2 mM each
    50 mM MgSO42 µl 2 mM
    Primer mix (10 µM each) 1 µl 0.2 µM each
    Template DNA  >  1 µl (as required)
    Platinum® Taq High Fidelity 0.2 µl 1.0 unit*
    Autoclaved, distilled water to 50 µl Not applicable

    *1.0 unit is sufficient for amplifying most targets. in some cases, more enzyme may be required (up to 2.5 units).
     
    If desired, a master mix can be prepared for multiple reactions, to minimize reagent loss and to enable accurate pipetting.
     
     
  3. Mix contents of the tubes and overlay with mineral or silicone oil, if necessary.

  4. Cap the tubes and centrifuge briefly to collect the contents.

  5. Incubate tubes in a thermal cycler at 94°C for 30 s to 2 min to completely denature the template and activate the enzyme.  Do not denaturate for more than 30 s if target is greater than 12 kb.

  6. Perform 25-35 cycles of PCR amplification as follows:
     
                            Denature        94°C for 15–30 s
                            Anneal           55°C for 15–30 s
                            Extend           68°C for 1 min per kb
      

  7. Maintain the reaction at 4°C after cycling.  The samples can be stored at -20°C until use.

  8. Analyze the products by agarose gel electrophoresis and visualize by ethidium bromide staining.  Use appropriate molecular weight standards.

Protocol with Platinum® Taq DNA Polymerase High Fidelity

Introduction

Platinum® Taq  DNA Polymerase High Fidelity is an enzyme mixture composed of recombinant Taq  DNA polymerase, Pyrococcus species GB-D polymerase, and Platinum® Taq Antibody.  Pyrococcus species GB-D polymerase possesses a proofreading ability by virtue of its 3´ to 5´ exonuclease activity.  Mixture of the proofreading enzyme with Taq  DNA polymerase increases fidelity approximately six times over that of Taq  DNA polymerase alone and allows amplification of simple and complex DNA templates over a large range of target sizes. Targets 12–20 kb can be amplified with some optimization. Targets greater than 20 kb require thorough optimization. The enzyme mixture is provided with an optimized buffer that improves enzyme fidelity and amplification of difficult templates.  

 
An anti-Taq  DNA polymerase antibody complexes with and inhibits polymerase activity.  Activity is restored after the denaturation step in PCR cycling at 94°C, thereby providing an automatic “hot start” for Taq DNA polymerase in PCR.  Hot starts in PCR provide increased sensitivity, specificity, and yield, while allowing assembly of reactions at room temperature. The use of this antibody helps reduce PCR optimization requirements, reaction set-up time and effort, handling of reaction components, and contamination risk, thereby improving PCR results.
 
Platinum® Taq  DNA Polymerase High Fidelity is supplied at the same 5 unit per µl concentration as Platinum® Taq  DNA Polymerase.  No modification to PCR reactions or protocols are necessary.


Kit Size
 
Component 100 rxn500 rxn 5,000 rxn
Platinum® Taq DNA Polymerase High Fidelity 20 µl 100 µl 1000 µl
10X High Fidelity PCR Buffer 1.25 ml 2.5 ml50 ml
50 mM Magnesium Sulfate 1 ml 1 ml25 ml

Platinum® Taq DNA Polymerase High Fidelity Storage Buffer

20 mM Tris-HCl (pH 8.0), 40 mM NaCl, 2 mM Sodium Phosphate, 0.1 mM EDTA, 1 mM DTT, stabilizers, and 50% (v/v) glycerol
 
10X High Fidelity PCR Buffer

600 mM Tris-SO4 (pH 8.9), 180 mM Ammonium Sulfate
 
Unit Definition

One unit of Platinum® Taq DNA Polymerase High Fidelity incorporates 10 nmol of deoxyribonucleotide into acid-preceptible material in 30 min at 74°C.
 
PCR Precautions

An extension temperature of 68°C, the use of thin-walled reaction tubes for targets above 5 kb, the use of one unit of enzyme for targets above 12 kb, and an increased primer concentration of 0.4 µM for plasmid and lambda targets are all recommended conditions.
 
Protocol

The following procedure is suggested as a guideline and starting point when using Platinum®Taq  DNA Polymerase High Fidelity in any PCR amplification. Reaction size may be altered to suit user preferences.
 
  1.     Add the following components to a autoclaved microcentrifuge tube at either ambient temperature, or on ice:

Components VolumeFinal Concentration
10X High Fidelity PCR Buffer 5 µl 1 X
10 mM dNTP mixture 1 µl 0.2 mM each
50 mM MgSO4  2 µl 2 mM
Primer mix (10 µM each)
1 µl
0.2 µM each
Template DNA
>  1 µl 
(as required)
Platinum® Taq  High Fidelity
 0.2 µl
1.0 unit*
Autoclaved, distilled water
to 50 µl
 Not applicable

*1.0 unit is sufficient for amplifying most targets. in some cases, more enzyme may be required (up to 2.5 units).
 
If desired, a master mix can be prepared for multiple reactions, to minimize reagent loss and to enable accurate pipetting.
 
  2.     Mix contents of the tubes and overlay with 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 30 s to 2 min to completely denature the template and activate the enzyme.  Do not denaturate for more than 30 s if target is greater than 12 kb.
 
  5.     Perform 25-35 cycles of PCR amplification as follows:
 
                        Denature       94°C for 15–30 s
                        Anneal          55°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 products by agarose gel electrophoresis and visualize by ethidium bromide staining.  Use appropriate molecular weight standards.

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Protocol with AccuPrime™ Taq High Fidelity

Use the following protocol as a starting point and guideline when preparing your reactions. Adjust the reaction size as needed. Use Buffer I for plasmids, cDNA, and l DNA and Buffer II for genomic DNA (up to 20 kb) as indicated.
 
  1.     Select a reaction mixture below based on your type of template. Add the components to a sterile, thin-walled 0.25-ml or 0.5-ml PCR tube either at room temperature or on ice. Note: For multiple reactions, we recommend a master mix to minimize reagent loss and enable accurate pipetting.

  Template Type
 
Component Plasmids/cDNA/l DNA Genomic DNA
10X AccuPrime™ PCR Buffer I 5 µl --
10X AccuPrime™ PCR Buffer II --5 µl
Sense primer (10 µM) 1 µl 1 µl
Anti-sense primer (10 µM) 1 µl 1 µl
Template DNA 0.1 pg–20 ng 10 pg–500 ng
AccuPrime™ Taq High Fidelity 0.2 µl* 0.2 µl*
Autoclaved, distilled water to 50 µl to 50 µl

*0.2 µl = 1.0 unit, which is sufficient for amplifying most targets. In some cases (e.g., longer targets), more enzyme may be required—up to 2.5 units.
 
  2.     Mix and overlay with 50 µl of mineral or silicone oil, if necessary.
 
  3.     Cap the tube and centrifuge briefly to collect the contents.
 
  4.     Incubate the tube in a thermal cycler at 94°C for 15 seconds to 2 minutes to completely denature the template and activate the enzyme.  Do not denature for more than 30 s if target is larger than 12 kb.
 
  5.     Perform 25-35 cycles of PCR amplification as follows:

                        Denature       94°C for 15–30 s
                        Anneal          52–64°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 products by agarose gel electrophoresis and visualize by ethidium bromide staining. Use appropriate molecular weight standards

References

  1.     Innis, M.A., Myambo, K.B., Gelfand, D.H. and Brow, M.A.D. (1988) Proc. Natl. Acad. Sci. USA 85, 9436.
 
  2.     Barnes, W.M. (1994) Proc. Natl. Acad. Sci. USA 91, 2216.
 
  3.     Tindall, K.R. and Kunkel, T.A. (1988) Biochemistry 27, 6008.
 
  4.     Chou, Q., Russel, M., Birch, D., Raymond, J., Bloch, W. (1992) Nucl. Acids Res., 20, 1717.
 
  5.     Sharkey, D.J., Scalice, E.R., Christy, K.G., Atwood, S.M., Daiss, J.L. (1994) BioTechnology, 12, 506.
 
  6.     Westfall, B., Sitaraman, K., Solus, J., Hughes, J., Rashtchian, A. (1997) Focus® 19, 46.
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