site directed mutagenesis
svetlov at oncology.wisc.edu
Thu Nov 12 11:48:35 EST 1998
In article <36496A6C.B9345561 at lg.ehu.es>, Oskar Fdz-Capetillo
<ggbferuo at lg.ehu.es> wrote:
> Hi there:
> I am just starting to develop my mutagenesis techniques. I am
> wondering about using the "quick change" Kit from Stratagene. I would
> want to know if anybody has used this kit and how did it work.
I was asked via email by several people about the protocol we use for
making QuickChange mutations and although it's quite unremarkable I'll post
it here in case anybody else is wondering.
The protocol is not much different from the standard QuickChange
described in Stratagene's Instruction manual (rev. #076002). The reaction
is set-up in 50 ul (total volume) with 5 ul of 10X reaction buffer (cloned
and native Pfu's from Strategene have somewhat different buffers so we
always use the cognate ones), ~200 ng of plasmid template, 20-40 pmoles of
each primer, 1 ul of 4X2.5 mM dNTPs mix and 1 ul (2.5U) of Pfu enzyme.
Reaction conditions are 1 min at 95, then 12 (for point mutations) to 25
(deletions) cycles (30 sec at 95, 1 min at 55, and 2 min for each kb of
template at 68 (e.g. 10 min for 5 kb plasmid). Reaction mix is then cooled
to 4 C and 10 units of DpnI is added directly to the reaction mix, mix by
pipetting and incubated at 37 C for 1 hour. Without heat inactivation or
any other processing 1 ul of the DpnI-treated reaction mix is added to the
50 ul of RbCl-treated competent cells (most often DH5alpha), transformation
carried out according to one's favorite routine.
Important note on the primer design: Tm (nearest neighbor) for the primer
should be 78C+ in case of primers introducing point mutation or a bulge of
2-3 mismatched mucleotides. In case of deletion/insertion primers with
substantial loop being introduced in either strand EACH HALF of the primer
(on either side of the introduced deletion/insertion) should have Tm of
70-75C for efficient mutagenesis. Thus on average my deletion primers are
Vladimir Svetlov, PhD
McArdle Lab for Cancer Res.
1400 University Ave.
Madison, WI 53706
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