Basic PCR program design

1. Initial denaturation for 2 minutes at 95⁰C
2. denature for 1 minute at 95⁰C
3. Anneal primers for 30 seconds at temperature ~5⁰C below melting temperature of primers.
4. Extend DNA at 72⁰C using each 1-2 minutes per kilobase of product, depending on whether you are using a polymerase with proofreading capabilities. If in doubt see manufacturers instructions.
5. Repeat steps 2-4 for 25-30 cycles.
6. Final extension for 10 min at 72⁰C
7. Cooling down to 4⁰C (usually time is set as eternity so it can be stored in the machine until you can take it out)