For a PCR with 25 Î¼l reaction volume add the following to your pcr master mix.
Prepare the following reaction mix on ice:
For a 25Î¼l reaction volume: Component Volume Final Conc. PCR Master Mix, 2X 12.5Î¼l 1X Forward Primer, 10Î¼M 0.25â€“2.5Î¼l 0.1â€“1.0Î¼M Reverse Primer, 10Î¼M 0.25â€“2.5Î¼l 0.1â€“1.0Î¼M DNA template 1â€“5Î¼l <250ng Nuclease-Free Water to 25Î¼l
A 2-minute initial denaturation step at 95Â°C.
(Subsequent denaturation steps can be between 30 seconds and 1 minute).
Step 2) Annealing
Approximately 5Â°C below the calculated melting temperature of the primers (Tm).
Usually 30 seconds to 1 minute.
Step 3) Extension
The extension reaction is performed at the optimal temperature for Taq
DNA Polymerase which is from 68â€“74Â°C depending on the Taq you are using (see manufacturers enzyme information).