Quick Change Mutagenesis

Hahn Lab 2001

This method uses the two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions (Wang and Malcolm, bioTechniques 26:680-682 (1999).

1. Set up two separate primer extension reactions (one for each top and bottom primer) containing:

5 microliters 10X Pfu Buffer (supplied with enzyme)
1 microliter 10 micromolar primer (0.13 microgram 45-mer)
0.1 – 0.2 microgram plasmid template
1 microliter 10 mM dNTP mix
H2O to a final volume of 50 microliters

Add 1 microliter Pfu turbo polymerase (Stratagene)

Incubate:

1. 94 deg, 30 sec
2. 95 deg, 30 sec
3. 55 deg, 1 min
4. 68 deg, 2 min/kb up to 10 KB plasmid

Repeat steps 2-4 for a total of 4 cycles
Hold at 4 deg.

Combine 25 microliters from each extension reaction above. Add 1 microliter Pfu polymerase. Incubate as above, except repeat a total of 18 cycles.

Remove 25 microliters of the reaction. Add 10 units of DpnI enzyme. Mix well and incubate at 37 deg for at least one hour.

Transform 1 microliter of the reaction to electrocompetent cells. Plate 100 and 20 microliters of cells on separate plates. Expect ~100 colonies on the 20 microliter cell plate.

Notes:

Expected mutagenesis frequency about 70%.

Stratagene recommends gel purifying the primers for the highest mutagenesis frequency, but not routinely done.

Primer pairs should have similar annealing temperatures. Melting temperatures between 55-80 deg are suggested.