Endy:Site-directed Mutagenesis

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Primer Design

  • See this page for general advice and software suggestions for primer design.
  • Primers should contain the mutations to be introduced but should anneal to the sequence to be mutated. You will need a forward and reverse primer.
  • Primers should be between 25 and 45 base-pairs in length.
  • The mutation(s) should be in the middle of the primers.
  • Mutation efficiency is greatly influenced by the purity of the primers. Its worth getting purified primers. No need for 5' phosphorylation.
  • See the Stratagene manual for more detailed information.

Experimental Protocol

  • See the Stratagene manual for the suggested protocols. The notes below are suggested modifications to this protocol that may be useful depending on what you are trying to do.
  • It makes sense to dilute the mutagenic primers to the same concentration as the control primers used in the Stratagene protocol, i.e. 100ng/μl.
  • Leon suggested that attempting to modify three consecutive bases is difficult. He suggested using 4-5 more cycles than recomended by Stratagene.
  • Leon also recommended letting the DpnI digestion run for 2-3 hours.
  • Doing a PNK step on the primers should boost efficiency.
  • It is not necessary to use XL1-Blue cells. Any highly competent cells should be ok.