1. After thawing, gently vortex and centrifuge all solutions.

2. Assemble all reaction components in PCR tubes on ice.

3. To prepare several reactions at once, create a master mix with all components except the template DNA. Then combine this with the DNA in each PCR tube.

4. Gently mix the reaction, using a quick spin to collect liquid if necessary. Overlay the mixture with 25µL of mineral oil when using a thermocycler without a lid.

5. Quickly transfer PCR tubes to the thermocycler pre-heated to the 95°C denaturation temperature. The recommended thermocycler conditions are as follows:

Initial denaturation: 95°C, 1 min (longer for GC-rich sequences)

25-40 cycles of denaturation (95°C, 30 secs), annealing (primer Tm, 45-68°C, 30 secs), extension (68°C, 1kb/min or ~5 mins)

Final extension: 68°C, 5 min

Hold: 4°C