This procedure is based on Kunkel, T. A. (1985). Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A 82, 488-492.
Particularly useful for plasmids larger than 8 kb.

The plasmid to be mutagenised has to have an f1 origin of replication for production of single stranded DNA. The plasmid is transformed in E. coli strain CJ236, which is deficient for dUTPase (dut) and uracil N-glycosilase (ung), thus producing ssDNA with high rate of incorporation of deoxy-uracil residues. This ssDNA is used as a template to anneal a phosphorylated mutant oligo, then an extension reaction is carried out in the presence of ligase. The produced dsDNA is transformed in XL1Blue or DH5alpha, which are capable of repairing the uracil-containing DNA strand, resulting in a plasmid carrying the desired mutation.

1. Transform plasmid in CJ236 and plate on LB/Chloramphenicol/antibiotic.

2. Use a single colony to make ssDNA (see additional protocol).

3. Phosphorylate mutant oligo (ideal oligo has >14 bp at each end of a point mutation).

200 pmols oligo 2 ul
10 x T4 Polynucleotide kinase buffer 2 ul
5 mM ATP 2 ul
T4-PNK (10 U/ul) 1 ul
------------------
20 ul. 1h 37oC. Store @ -20oC.

4. Anneal phosphorylated primer to ssDNA @ molar ratio ~ 20:1. For example

5 pmols oligo
500 ng ssDNA
1 ul 10 x ligase buffer
-----------
10 ul.
Denature 5 min 94oC. Drop temperature to 70oC and decrease by 1oC/min, till 30oC. Store reaction @ -20oC.

5. Extension

Annealing reaction 10 ul
1.25 mM dNTPs 2 ul
10 x ligase buffer 1 ul
Klenow, 2.5 U/l 1 ul
Ligase, 400 U/l 1 ul
H2O 5 ul
---------------------
20 ul . 1h RT
Transform E. coli, plate the whole reaction.