Site-directed mutagenesis

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Notes)
Line 3: Line 3:
==General Procedure==
==General Procedure==
 +
#Purify template plasmid DNA from a dam<sup>+</sup> ''Escherichia coli'' strain (to ensure that all GATC sites are methylated for later digestion with DpnI).
#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.
#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.
#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.
#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.

Revision as of 06:42, 16 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 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.

References

Personal tools