Editing 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 10Ã reaction buffer<br>X ÎŒl (5â50 ng) of dsDNA template<br>X ÎŒl (125 ng) of oligonucleotide F primer #1<br>X ÎŒl (125 ng) of oligonucleotide R primer #2<br>1 ÎŒl of dNTP mix<br>ddH2O to a final volume of 25 ÎŒl<br>Then add 0.5 ÎŒl of PfuTurbo DNA polymerase (2.5 U/ÎŒl)

=== PCR<br> ===

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

[[Category:Protocols]]