Richard Lab:Site Directed Mutagenesis

From OpenWetWare

Revision as of 17:51, 25 January 2011 by Michael A. Speer (Talk | contribs)
Jump to: navigation, search

Back to Protocols

Site Directed Mutagenesis

Contents

Introduction

This protocol is used if you want to change one to three consecutive bases in a sequence (like removing a restriction site or changing an amino acid. It utilizes a fast high-fidelity non-displacing DNA polymerase (Phusion) to replicate the plasmid including the desired mutation. DpnI then cuts up all your old plasmid.

Procedure

1. Design mutagenesis primers.

  • The targeted mutation should be included into both primers.
  • The mutation can be as close as 4 bases from the 5-terminus.
  • The mutation should be at least 8 bases from the 3-terminus.
  • At least eight non-overlapping bases should be introduced at the 3-end of each primer.
  • At least one G or C should be at the end of each primer.
  • Design your primers to have a melting temperature >=78°C.

2. Purify template plasmid from a dam+ E. coli strain via miniprep. 3. Set up mutagenesis PCR mix

  • 36µl water
  • 10µl 5X Phusion Buffer
  • 1µl dNTPs (25mM each)
  • 1µl Primer F
  • 1µl Primer R
  • 0.5µl Template DNA
  • 0.5µl Phusion Polymerase

4. Run PCR

  1. 98°C for 30 secs
  2. Run the following for 20 cycles:
    1. 98°C for 10 secs
    2. 60°C for 30 min
    3. 72°C for 30 sec/kb of plasmid length minimum
  3. 72°C for 5 mins
  4. 4°C infinite

5. Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary) 6. Incubate 2-3 hours at 37°C. 7. Purify PCR product and elute into 30μL. 8. Transform 3μL purified DNA into highly competent cells. 9. Screen the transformants for the desired mutation using restriction digest or sequencing

Notes

  • In Mike's capable hands 70% of colonies are correct using this protocol
  • Just pick two colonies to sequence and at least one of them will work.

references

Zheng, L., U. Baumann, and Jean-Louis Reymond. 2004. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res. 2004; 32(14): e115.

Back to Protocols

Personal tools