Endy:Site-directed Mutagenesis

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(Experimental Protocol)
Current revision (07:52, 24 August 2005) (view source)
 
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''Back to [[Site-directed mutagenesis]]''
==Primer Design==
==Primer Design==
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*See [[Designing Primers | this page]] for general advice and software suggestions for primer design.
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*See [[Designing primers | 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 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.
*Primers should be between 25 and 45 base-pairs in length.
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==Experimental Protocol==
==Experimental Protocol==
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*See the [http://www.stratagene.com/manuals/200518.pdf Stratagene manual] for the suggested protocols.
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*See the [http://www.stratagene.com/manuals/200518.pdf 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.
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*Comments and usage notes on this protocol will be added when the protocol has been tested.
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*It makes sense to dilute the mutagenic primers to the same concentration as the control primers used in the [http://www.stratagene.com Stratagene] protocol, i.e. 100ng/μl.
*It makes sense to dilute the mutagenic primers to the same concentration as the control primers used in the [http://www.stratagene.com 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 [http://www.stratagene.com Stratagene].
*Leon suggested that attempting to modify three consecutive bases is difficult.  He suggested using 4-5 more cycles than recomended by [http://www.stratagene.com Stratagene].
*Leon also recommended letting the DpnI digestion run for 2-3 hours.   
*Leon also recommended letting the DpnI digestion run for 2-3 hours.   
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*Doing a [[PNK Treatment of DNA Ends|PNK step]] on the primers should boost performance.
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*Doing a [[PNK Treatment of DNA Ends|PNK step]] on the primers should boost efficiency.
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*It is not necessary to use XL1-Blue cells.  Any highly competent cells should be ok.

Current revision

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.
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