Introduction

Platinum® GenoTYPE Tsp DNA Polymerase is a recombinant DNA polymerase from a thermophilic species of bacteria. It is intended for use in genotyping of dinucleotide repeat loci. The polymerase has been engineered to lack both 5´ and 3´ exonuclease activities and is severely restricted in its ability to add a nontemplated nucleotide to the end of the PCR product. It can be substituted directly for Taq  DNA polymerase in amplification reactions as a simple solution to the heterogenous extra nucleotide addition problem.

It is recommended for amplification of fragments up to 500 bp in length. The enzyme is supplied complexed with proprietary antibody that inhibits polymerase activity. Due to specific binding of the inhibitor, Platinum® GenoTYPE Tsp DNA Polymerase is provided in an inactive form.  This reagent provides an automatic “hot start” for use in PCR(1,2).  Hot starts are typically used in PCR to increase sensitivity, specificity and yield while allowing assembly of reactions at ambient temperatures.  The extra time, effort, and contamination risks associated with manual hot start procedures are addressed with the use of Platinum® GenoTYPE Tsp DNA Polymerase. The activity of Platinum® GenoTYPE Tsp DNA Polymerase is blocked at ambient temperatures but is regained after the denaturation step in PCR cycling at 94°C.
 
Components:

Platinum® GenoTYPE Tsp DNA Polymerase
10X PCR Buffer, Minus Mg
50 mM Magnesium Chloride
 
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
 
Unit Definition: 

One Tsp unit of Platinum® GenoTYPE Tsp DNA Polymerase has been functionally determined to be equivalent to one unit of Taq DNA Polymerase in amplification of dinucleotide repeats using standard Taq reaction conditions. One Tsp unit approximates 2.5 activity units. An activity unit incorporates 10 nmol of deoxyribonucleotide into acid-precipitable material in 30 min at 74°C under optimized reaction conditions.
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Ordering Information

Sku Name Size Price Qty
11448024 Platinum® GenoType Tsp DNA Polymerase 250 units USD 349.00
11448032 Platinum® GenoType Tsp DNA Polymerase 2500 units USD 2,525.00

Protocol

The following general procedure is suggested as a guideline and as a starting point when using Platinum® GenoTYPE Tsp DNA Polymerase in any PCR amplification.
 
  1.     Add the following components to the PCR reaction tube:
 
Components Volume Final Concentration
10X PCR Buffer, Minus Mg1.5 µl1X
10 mM dNTP mixture0.3 µl0.2 mM each
50 mM MgCl2 0.45 µl1.5 mM
Primer mix (5 µM each)1 µl0.33 µM each
Template DNA as required 50 ng
Platinum®GenoTYPE Tsp 0.12 µl0.6 units
DNA Polymerase  
Autoclaved, distilled waterto 15 µlNot applicable

 
If desired, a master mix can be prepared for multiple reactions, to minimize reagent loss and to enable accurate pipetting.
 
  2.     Perform 30 cycles of PCR amplification as follows:
 
Predenaturation  94°C for 1-2 min (if desired)
    
     10 cycles of:
                   Denature       94°C for 30 s
                   Anneal          55°C for 30 s
                   Extend          72°C for 1 min
    
     20 cycles of:
                   Denature       89°C for 30 s
                   Anneal          55°C for 30 s
                   Extend          72°C for 1 min
      Final extension          72°C for 10 min (if desired)
 
  3.     Maintain the reaction at 4°C after cycling.  The samples can be stored at -20°C until use.
 
  4.     Analyze the amplification products by electrophoresis.  Use appropriate molecular weight standards to determine the size of the products.
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