Designing primers

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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)
  • Molecular Cloning advises GC content 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