Richard Lab:Site Directed Mutagenesis

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Site Directed Mutagenesis

  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.
  1. . Purify template plasmid from a dam+ E. coli strain via miniprep.
  2. . 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. 98°C for 10 secs 3. 60°C for 30 min 4. 72°C for 30 sec/kb of plasmid length minimum 5. Run PCR for 20 cycles 6. 72°C for 5 mins 7. 4°C Add 1μL DpnI restriction enzyme to the PCR tube directly. (Purification is not necessary at this stage). Incubate 2-3 hours at 37°C. Purify PCR product. Transform purified DNA into highly competent cells. Screen the transformants for the desired mutation using restriction digest or sequencing as appropriate. Typically 1/4 or 1/8 colonies are correct.