Difference between revisions of "Designing primers"

From OpenWetWare
(Useful Primer Design Tools)
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*[https://catalog.invitrogen.com/index.cfm Invitrogen]
*[https://catalog.invitrogen.com/index.cfm Invitrogen]
*[http://frodo.wi.mit.edu/primer3/primer3_code.html Primer3]
*[http://frodo.wi.mit.edu/primer3/primer3_code.html Primer3]
*[http://synbio.mit.edu/tools/clipboard.cgi Austin's clipboard tool] - online tool for generating the complement, reverse complement and restriction enzyme site analysis of a DNA sequence.  It also translates the sequence and gives the amino acids properties.

Revision as of 19:27, 30 May 2005

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.
  • Avoid 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