Richard Lab:Site Directed Mutagenesis

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