Richard Lab:Site Directed Mutagenesis
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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.
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
- 98°C for 30 secs
- Run the following for 20 cycles:
- 98°C for 10 secs
- 60°C for 30 min
- 72°C for 30 sec/kb of plasmid length minimum
- 72°C for 5 mins
- 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
- 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.
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.
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