Richard Lab:Site Directed Mutagenesis

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Site Directed Mutagenesis

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

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