Designing primers

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This needs more work, 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