Site-directed mutagenesis

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(Specific Protocols)
(Notes)
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*Although the manual recommends only 12 cycles for point mutations, I usually do more (at least 18 cycles). --[[User:Rshetty|Reshma]] 05:28, 14 Jul 2005 (EDT)
*Although the manual recommends only 12 cycles for point mutations, I usually do more (at least 18 cycles). --[[User:Rshetty|Reshma]] 05:28, 14 Jul 2005 (EDT)
*I tried using this protocol to do an insertion/deletion and I believe it failed suggesting that point mutations are easier to accomplish than insertions/deletions. --[[User:Rshetty|Reshma]] 05:28, 14 Jul 2005 (EDT)
*I tried using this protocol to do an insertion/deletion and I believe it failed suggesting that point mutations are easier to accomplish than insertions/deletions. --[[User:Rshetty|Reshma]] 05:28, 14 Jul 2005 (EDT)
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*[[User:Lchan|Leon Chan]] 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].
+
*[[User:Lchan|Leon Chan]] suggested that attempting to modify three consecutive bases is difficult.  He suggested using 4-5 more cycles than recommended by [http://www.stratagene.com Stratagene].
*[[User:Lchan|Leon]] also recommended letting the DpnI digestion run for 2-3 hours.   
*[[User:Lchan|Leon]] also recommended letting the DpnI digestion run for 2-3 hours.   
*Doing a [[PNK Treatment of DNA Ends|PNK step]] on the primers should boost efficiency.
*Doing a [[PNK Treatment of DNA Ends|PNK step]] on the primers should boost efficiency.
*It is not necessary to use XL1-Blue cells.  Any highly competent cells should be ok.
*It is not necessary to use XL1-Blue cells.  Any highly competent cells should be ok.
 +
*Stratagene recommends trying various concentrations of template DNA in the PCR step.
==References==
==References==
*[[Molecular Cloning]]
*[[Molecular Cloning]]
*[http://www.stratagene.com/manuals/200518.pdf Stratagene QuikChange Site-directed Mutagenesis Kit]
*[http://www.stratagene.com/manuals/200518.pdf Stratagene QuikChange Site-directed Mutagenesis Kit]

Revision as of 08:51, 24 August 2005

Contents

General Information

Site-directed mutagenesis can be used to change particular base pairs in a piece of DNA. There are a number of methods for achieving this. The approach described here is adapted from the Stratagene site-directed mutagenesis kit, the manual can be found here. Even when using a kit it will be necessary to design primers that are suitable for the specific changes you want to make to your DNA. Most of the contents of the kit can be found in your favorite labs stocks so you may not need to buy the kit itself.

General Procedure

  1. Purify template plasmid DNA from a dam+ Escherichia coli strain (to ensure that all GATC sites are methylated for later digestion with DpnI).
  2. Design forward and reverse primers that will bind to the region of DNA you want to mutate but that contain the modifications you wish to make.
  3. Run a PCR reaction with a proof-reading, non-displacing polymerase such as PFU DNA polymerase. This results in nicked circular strands of the plasmid.
  4. Cut up the template DNA with DpnI.
  5. Transform the circular nicked DNA into a highly competent strain such as XL1-Blue. These cells will repair the nicks.
  6. Select colonies with the correct DNA.

Specific Protocols

Notes

  • This protocol is at a very early stage of development. Any contributions welcome!
  • Although the manual recommends only 12 cycles for point mutations, I usually do more (at least 18 cycles). --Reshma 05:28, 14 Jul 2005 (EDT)
  • I tried using this protocol to do an insertion/deletion and I believe it failed suggesting that point mutations are easier to accomplish than insertions/deletions. --Reshma 05:28, 14 Jul 2005 (EDT)
  • Leon Chan suggested that attempting to modify three consecutive bases is difficult. He suggested using 4-5 more cycles than recommended 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.
  • Stratagene recommends trying various concentrations of template DNA in the PCR step.

References

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