This needs more work, but wanted to get it started.
- Avoid runs over 3 nucleotide (AAAA)
- 18-30bp in length. Molecular Cloning says that 5' tails do not significantly affect annealing.
- Primer pairs should differ in length by less than 3bp.
- 3’ end should be G or C (stronger bond)
- primer melting temp (Tm) should be 50-60C w/ low FIR difference (<5C, 2C better)
- 30-80% GC (>50% is better) (Molecular Cloning advises between 40% and 60%)
- avoid palindromes and inverted repeat sequences.
- Avod complementarity between members of a primer pair.
- check for dimer binding and hairpins in Vector NTI.
- want to avoid structures with ΔG < -5kcal/mol
Useful Primer Design Tools