Designing Primers – A Checklist

Do annealing temperature of each primer pair PCR products corresponding to the same?

Whether primary has
annealing segment?
segment tags?
restriction enzyme sites?
Extra bases to help cut restriction sites?

Designing Primers – A Checklist

The primary is in the correct orientation (if necessary, it in reverse complement? If necessary, is extra base [d] in a complementary way around?)

Annealing temperature is at least 50 degrees Celsius?