Site Directed Mutagenesis Protocol

Protocol:

1. Primer design:
   • Design primers containing the desired mutation in the center. The mutation should be flanked by 12-18 bases on both sides. Use online tools to check primer specificity and avoid unintended mutations.
2. PCR amplification:
   • Perform a PCR reaction using the plasmid template, forward and reverse primers, high-fidelity DNA polymerase, and dNTP mix. Use the following PCR conditions:
     • Initial denaturation: 95°C for 2 minutes
     • Denaturation: 95°C for 30 seconds
     • Annealing: (Use Tm of primers – 5°C) for 30 seconds
     • Extension: 68°C for 1 minute/kb of plasmid length
     • Repeat steps 2-4 for 18-30 cycles
     • Final extension: 68°C for 5 minutes
3. DpnI digestion:
   • Add DpnI directly to the PCR reaction mixture to digest the methylated parental DNA template. Incubate at 37°C for 1-2 hours.
4. Transformation:
   • Transform chemically competent E. coli cells with the DpnI-digested PCR product. Follow the manufacturer’s instructions for transformation.
5. Plating and Selection:
   • Plate the transformed cells on LB agar plates containing the appropriate antibiotic. Incubate overnight at 37°C.
6. Colony Screening:
   • Select several colonies and perform colony PCR to confirm the presence of the desired mutation. Sequence the positive colonies to validate the mutation.