Download `Quickie` Quikchange mutagenesis (Stratagene)

‘Quickie’ Quikchange mutagenesis (Stratagene)
O. Gjoerup 12-1-99
1) Set up the following reaction on ice:
1 µl = 15 ng template (5-50 ng)
1 µl = 125 ng oligo 1
1 µl=125 ng oligo 2 (complementary to 1)
1 µl dNTP mix
5 µl 10x reaction buffer
41 µl dH20 (5 µl total volume)
Finally, add 1 µl Turbo Pfu DNA polymerase (2.5 U/µl)
2) Thermo-cycle as follows:
95 ˚C
30 s
(1 cycle)
(95 ˚C 30 s, 55 ˚C 1 min, 68 ˚C 2 min./ kb of template) (12-18 cycles)
(extension time will be long because the template that needs to be extended is large)
3) When samples have cooled down, add 1 µl DpnI and incubate at 37 ˚C for 1 h.
4) Use 4 µl of the reaction mix for a transformation into highly competent cells, e.g.
100 µl of Promega’s JM109 (cat# L2001, 5x 200 µl aliquots) competent cells.
Things to consider:
a) The most frequent problem, not surprisingly, is ‘no colonies’. Almost always, if you
get colonies (even just a couple), you’re going to get your mutant. To facilitate screening
for mutants, you may incorporate a ‘silent’ (one that doesn’t change the coding)
restriction site (or aim to lose a site). However, ultimately you want to sequence the
construct.
b) If you do get zero colonies you can try to ethanol precipiate the whole reaction and
transform everything. Alternatively, you can increase the number of cycles, or the
amount of template DNA. Control your reactions ! at least the first few times. Make
sure the problem is not with the competent cells – Quikchange usually works very well
but requires highly competent cells. Use a small amount (say 1 pg) DNA as a
transformation control (it comes with the JM109 competent cells from Promega). Also,
do the mutagenesis control included with the kit; this will tell you if you’re experiencing
a problem related to the reagents or the specific primer set.