Site-Directed Mutagenesis Protocol using PCR

Biochemistry, Molecular Biology, and Cell Biology Protocols >> PCR Site-Directed Mutagenesis Protocol

1. Design a pair of primers such that the mutation(s) is near the middle of the primer, and the primer has a sufficiently high melting temperature
2. Perform 12-16 rounds of PCR on the entire vector
(Include a control sample where you only incubate with one primer, with water replacing the other primer.)
3. Check the PCR product on a gel; if there is no product, lower the PCR annealing temperature; if there is a smear, increase the PCR annealing temperature
4. Digest with DpnI for 2 hours at 37 degrees C.
5. Transform into competent cells, and plate onto agar plates.
6. Pick colonies and grow up the bacteria overnight in cultures.
7. Purify the plasmid, check the concentration, and sequence the DNA of the insert to make sure that the desired mutation has occurred, and no other random mutations are present.
8. Express the protein of interest via transfection in mammalian cells or other methods.