PCR and colony PCR

PCR

Phusion Hot Start II DNA Polymerase is used.
Prepare the 50 μL PCR reaction:

10 μL 5X Phusion HF Buffer
1 μL 10 mM dNTPs
5 μL of 5 μM Primer 1 and 2
1 μL of Template DNA
Water up to 50 μL

Run PCR, protocol depends on the length of the fragment and primer annealing temperatures. If the PCR product will be used for further reactions purify it using Thermo Scientific™ GeneJET PCR Purification Kit according to manufacturers instructions.

Colony-PCR

If colony PCR is run, use the same protocol, but instead of DNA template, pick a colony with a sterile toothpick, streak on a new LB-agar plate, and dip the same toothpick into the reaction mix just before starting PCR reaction.

Run PCR reactions on agarose gels to confirm the length of the amplified DNA fragments.