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The following protocol uses the DyNAzyme EXT DNA Polymerase. The conditions
used are the suggested optimal for 0-10 kb fragments. For longer fragments (up to 40 kb), check
the DyNAzyme EXT DNA Polymerase product guide.
1. Setup the following 50 ml PCR reaction:
| 10X DyNAZyme EXT PCR Reaction Buffer |
5 ml |
| Forward primer (20 mM, 20 pmol/ml ) |
1 ml |
| Reverse primer (20 mM, 20 pmol/ml ) |
1 ml |
| 10 mM dNTP (mix of 10 mM dATP, dCTP, dGTP, dTTP each) |
1 ml |
| DyNAzyme EXT DNA Polymerase (1 units/ml) |
1 ml |
| DNA template (20-200 ng/ml or diluted bacteria culture) |
1 ml |
| ddH2O |
40 ml |
| Total volume |
50 ml |
2. Run PCR using the following program:
| Step 1 |
Denaturation 94 °C |
2 minutes |
| Step 2 |
Denaturation 94 °C |
45 seconds |
| Step 3 |
Annealing x(45-65) °C |
45 seconds |
| Step 4 |
Extension 72 °C |
1 minute/1.3-1.5 kb |
| Step 5 |
Repeat step 2-4 29 times |
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| Step 6 |
Extension 72 °C |
5 minutes |
| Step 7 |
Storage 4 °C |
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