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PCR --- long products

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
Step 6 Extension 72 C 5 minutes
Step 7 Storage 4 C