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             11448-024
10X PCR Buffer, Minus Mg                                           Y02028
50 mM Magnesium Chloride                                          Y02016

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.

Product Qualification

The Certificate of Analysis (CofA) provides detailed quality control information for each product. The CofA is available here, and is searchable by product lot number, which is printed on each box.

Ordering Information

Catalog # Name Size List Price (USD) Qty
11448024 Platinum® GenoType Tsp DNA Polymerase 250 units 349.00

Protocol

The following basic protocol serves as a general guideline and a starting point. It is designed to test varying concentrations (0X to 4X) of PCRx Enhancer Solution.

  1. Add the following components to the PCR reaction tube:

    ComponentVolumeFinal Concentration
    10X PCR Buffer, Minus Mg
    1.5 μl
    1X
    10 mM dNTP mixture
    0.3 μl
    0.2 mM each
    50 mM MgCl2
    0.45 μl
    1.5 mM
    Primer mix (5 μM each)
    1 μl
    0.33 μM each
    Template DNA
    as required
    50 ng
    Platinum® GenoTYPE Tsp
    DNA Polymerase
    0.12 μl
    0.6 units
    Autoclaved, distilled water
    to 15 μl
    Not 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)
    Denature 
    94°C for 30 s
    Anneal
    55°C for 30 s
    Extend
    72°C for 1 min for 10 cycles
    Denature
    89°C for 30 s
    Anneal
    55°C for 30 s
    Extend
    72°C for 1 min for 20 cycles
    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.

    TOP

References

  1. Chou, Q., et al. (1992) Nucl. Acids Res., 20, 1717.

  2. Sharkey, D.J., et al. (1994) BioTechnology, 12, 506.
MAN0000982      7-Jun-2010