Site Directed Mutagenesis (QuickChangeTM)

According to Wang and Malcolm, 1999 and Stratagene ( La Jolla, CA, USA)

Design primers that flank the mutation site for about 15 bp in the 5’ and 3’ direction. So the usual primer size is about 33-37 base pairs (bp). The reverse primer is designed by simply reverse complementing the forward primer.
Note: If possible, primersshould be designedsuch that a restriction enzyme recognition site is created or deleted in the mutant compared to the wild type sequence. This way it is easier to check whether the mutation was created. It is preferable to change as few bases as possible to create a certain amino acid exchange.
As both primers fit to each other better than to the template, run the first 5 cycles with the primers separately. Set up a second set of reactions without polymerase.
Note: The elongation time has to be chosenso that the whole templateplasmid can be amplified (depending on the polymerase and the plasmidsize).
Take 25 µl of each reaction and mix (A+B and C+D). Add more polymerase to the A+B reaction and allow the combined reaction to proceed for 13 cycles.
After the PCR reaction, digest 10 µl of PCR reactions with 0.5 µl DpnI (in the DpnI buffer) for 1 hour at 37 °C.
Use 5 µl of the digest to transform E.coli cells.
Grow transformed cells on appropriate antibiotic plates overnight at 37°C.
Pick several colonies and inoculate liquid cultures. Grow over night at 37 °C.
Mini prep the plasmids and confirm mutations by restriction enzyme digest and sequencing.

PCR reactions. A, B, C, D

	50 μl reaction	Final concentration
Template plasmid (~50 ng)	xx
Polymerase buffer 5X Buffer	10 μl
dNTPs (10 mM)	1 μl	200 μM
Primer (10 µM)	2.5 μl	0.5 μM
Polymerase	0.5 μl
Water	xx
	50 μl

A = fwd Primer
B = rev Primer
C = fwd Primer WITHOUT Polymerase
D = rev Primer WITHOUT Polymerase

References

Wang, W. and Malcolm, B. A. (1999). Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange Site-Directed Mutagenesis. Biotechniques 26, 680–682.