Designing primers
From OpenWetWare
This needs more owrk, but wanted to get it started.
Some general rules:
- Avoid runs over 3 nucleotide (AAAA)
- 18-30bp in length (what about tails?)
- 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)
- avoid palindromes
- check for dimer binding and hairpins in Vector NTI
- want to avoid structures with ΔG < -5kcal/mol