| Description: |
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GC-rich PCR DNA Polymerase is a high fidelity thermostable polymerase that amplifies target DNA up to 6 kbp in length with superior accuracy and yield. The enzyme’s 3´→5´ exonuclease-dependent proofreading activity results in a lower PCR mutation frequency than any other commercially available DNA polymerase. The elongation rate and processivity are 5 times and 10 to 15 times higher, respectively, than for Pfu DNA polymerase, resulting in highly accurate and robust yields in short reaction times. The enzyme has no 5´→3´ exonuclease activity and generates blunt-ended PCR products suitable for cloning.
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| Features: |
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- PCR amplification of DNA fragments as long as 6 kb, 30 sec/kb
- Eight times more accurate than Taq DNA polymerase.
- Highly thermostable - remains 95% active after 2 hours incubation at 95°C.
- Generates blunt-end PCR products. |
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| Concentration: |
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5 u/μl |
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| Storage conditions: |
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50 mM Tris-HCl (pH 8.0); 50 mM KCl; 1 mM DTT; 0.1 mM EDTA; stabilizers; and 50% (v/v) glycerol
Store at -20°C |
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| Reaction buffer GC-rich (10x): |
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1.2 M Tris-HCl (pH 8.0); 100 mM KCl; 60 mM (NH4)2SO4; 1% Triton X-100; 0.01% BSA
extra: 25 mM MgCl2 |
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| Unit definition: |
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One unit is defined as the amount of enzyme that will catalyze the incorporation of 10 nmol dNTP into acid insoluble form in 30 minutes at 75°C in a reaction containing 20 mM Tris-HCl (pH 7.5 at 25°C), 8 mM MgCl2, 0.5 mM DTT, 50 μg/ml BSA, 150 μM each of dATP, dCTP, dGTP, dTTP (a mix of unlabeled and [3H] dTTP) and 150 μg/ml activated calf thymus DNA.
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| Sample protocols |
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In many cases, the standard reactions described below will provide satisfactory amplification. Remember to include a negative control reaction lacking only template; inclusion of a positive control reaction using a template known to amplify with the primers may also be helpful. Concentrations of enzyme, MgCl2, template and primers can be varied to optimize the reaction.
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39μl
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H2O |
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1 μl
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template DNA |
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0.5 μl
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3´ primer (concentration 100pmol/μl) |
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0.5 μl
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5´ primer (concentration 100pmol/μl) |
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1 μl
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dNTP-Mix (10 mM each) |
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5 μl
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10x Reaction buffer C |
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2 μl
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25 mM MgCl2 (final concentration 1 mM) |
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0.5-1.0 μl
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GC-rich PCR DNA Polymerase (5 u/μl) |
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- Always mix on ice!
- The addition of 2-5% DMSO can improve amplification with GC-rich or longer templates and will not decrease the fidelity.
- Mix gently and centrifuge to bring reaction components to the bottom of the tube.
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Sample program for genomic template DNA up to 2 kbp: |
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| First Denature: |
5 min 95°C |
| Denature: |
15-30 sec 95°C |
| Anneal: |
30 sec (Tm-5)°C |
| Extend: |
35-65 sec/1kbp 72°C |
| Cycles: |
30 |
| Final extension: |
5 min 72°C |
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| Extension time: |
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Since GC-rich PCR DNA Polymerase is highly processive, long extension times may cause smearing. A two step cycling reaction, that combines the annealing and extension steps, is often used for short target DNA from template DNA in multiple copies (plasmid and phage DNA). Although the extension time can be increased for longer target DNA, targets above 6 kbp are difficult to amplify due to the strong 3´→5´ exonuclease activity.
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| Extension temperature: |
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The extension temperature can be increased to 74°C to increase yield. |
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| PCR protocols and reaction conditions have to be optimized for every individual template! |
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Long PCR amplifications are very sensitive to even small variations in the reaction conditions.
Therefore, the optimal conditions must be determined experimentally. |
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