Designing primers

This needs more work, but wanted to get it started.

General Rules

• 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

• VectorNTI
• Invitrogen