Richard Lab:Site Directed Mutagenesis

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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

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

references

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