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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.
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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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:
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.
1. Add the following components to the PCR reaction tube:
| Components | Volume | Final 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 | 0.12 µl | 0.6 units |
| DNA Polymerase | ||
| 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)
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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