Qualitative Verification of Target Sequence
Using Platinum Taq DNA Polymerase

Platinum Taq DNA Polymerase is recombinant Taq DNA polymerase complexed with a proprietary antibody that blocks polymerase activity at ambient temperatures. 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(1,2,3). Hot starts in PCR enable 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.

Platinum Taq DNA Polymerase is supplied at the same 5 unit per µl concentration as Taq DNA Polymerase (Cat. No. 10342-053). No modification to PCR reactions or protocols are necessary. This enzyme formulation can also be used in larger volume cocktail mixes without difficulty.

Storage Buffer

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

200 mM Tris-HCl (pH 8.4), 500 mM KCl
 
Quality Control Platinum Taq DNA Polymerase is evaluated in a DNA polymerization activity assay that measures the percent of Taq DNA polymerase inhibition versus an uninhibited control. Platinum Taq DNA Polymerase is functionally tested for amplification and the absence of double- and single-stranded endonuclease activity, as well as the absence of contaminating exonuclease activity.

Unit definition

One unit of Platinum Taq DNA Polymerase incorporates 10 nmol of deoxyribonucleotide into acid-precipitable material in 30 min at 74°C.
 
PCR precautions

Since PCR is a powerful technique capable of amplifying trace amounts of DNA, 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. Avoid contamination with the primers or template DNA used in individual reactions.  Analyze PCR products in an area separate from the reaction assembly area.

The procedure on the following page is suggested as a guideline and starting point when using Platinum Taq DNA Polymerase in any PCR amplification. Optimal reaction conditions (incubation times and temperatures, concentration of Platinum Taq  DNA Polymerase, primers, MgCl₂, and template DNA) vary and need to be optimized. Reaction size may be altered to suit user preferences.                                                                                              

  1.    Add the following components to a sterile 0.5-ml microcentrifuge tube:

*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 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 30 s to 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 30 s
Anneal                  55°C for 30 s
Extend                  72°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