from Grace Gill via Betsy O'Neill

SITE-DIRECTED MUTAGENESIS

site-dirested mutagenesis;
Use of the strain CJ236; (ung- dut-)to make the single stranded template DNA site-directed mutagenesis causes dUTP to be incorporated in place of dTTP. Later in the procedure after the mutation containing primer is annealed, the second strand is synthesized in vitro and the plasmid is transformed into TG-2; cells, the repair mechanisms in the cell tend to repair the uracil-containing parental strand, thus increasing the frequency of appearance of the desired mutant. Reports indicate an increase from about 5% to about 70% (Biotechniques vol. 5, pp786-791, 1987).

1) Purify oligo by TLC or electrophoresis.
2) Kinase 50 pmole of oligo in a 25µl reaction.
3) Set up 2 annealing reactions, one with primer and one without primer.
ssDNA 1µl (see other protocol)
primer 1 or 0 µl (2pm kinased oligo)
10x anneal 1 µl
ddH2O to 10 µl
4) Heat to 70°C/10 minutes. Cool slowly to 30°C, then place on ice.
5) For extension reaction, add to primer annealed template:
10x extens 2µl
dNTPs 4µl (2.5mM each)
ligase 1µl (dilute concentrated NEB stuff 4x)
T4 Pol 1µl
ddH2O 2µl
6) Leave on ice 5 minutes. Transfer to RT 5 minutes. Incubate at 30°C/90 minutes. Lower temperatures may be necessary for primers with a lot of mismatches.
7) Bring volume to 100ul with TE. Phenol/cholroform extract, ethanol precipitate.
8) Transform 1/10th of reaction into TG2.

10x Annealing Buffer 10x Extension Buffer
200 mM Tris, pH 7.5 10mM ATP
20 mM MgCl2 100 mM Tris, pH7.5
500 mM NaCl 50 mM MgCl2
20 mM DTT