Site-Directed Mutagenesis

Oligo Design and Synthesis

Synthesize two complimentary oligonucleotides containing the desired mutation, flanked by unmodified nucleotide sequence (10 to 15 bp).

PCR

Mix

2.5 ÎŒl of 10× reaction buffer
X ÎŒl (5–50 ng) of dsDNA template
X ÎŒl (125 ng) of oligonucleotide F primer #1
X ÎŒl (125 ng) of oligonucleotide R primer #2
1 ÎŒl of dNTP mix
ddH2O to a final volume of 25 ÎŒl
Then add 0.5 ÎŒl of PfuTurbo DNA polymerase (2.5 U/ÎŒl)

PCR

1. 95°C 30 seconds
2. 95°C 30 seconds
3. 55°C 1 min
4. 68°C 2 minutes/kb of plasmid length
cycles:
Point mutations 12
Single amino acid changes 16
Multiple amino acid deletions or insertions 18
5. Cool to RT

Digestion

Add 1 ÎŒl of the Dpn I restriction enzyme (10 U/ÎŒl) digest at 37°C for 1 hour

Transformation

Transform Top10/STBL3 with 5 to 10 ÎŒl digested reaction mix.

Plate on LB/Amp or LB/Kan