Endy:Site-directed Mutagenesis

Back to Site-directed mutagenesis

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.