QuikChange II Site-Directed Mutagenesis
hzhen at freeuk.com
Sat Oct 22 12:23:38 EST 2005
> I agree. We also noticed that our success rate is better when we go
> down from what was originally recommended by Stratagene.
> Actually, now that I realize that QuikChange II is simply good
> old QuikChange with PfuUltra (which means I've been using version
> II before Stratagene started to make it!), I do have what I'd consider
> the most useful trick in difficult cases:
> Do few cycles (1-3) with just one of the primers, then mix the two
> reactions and continue as normal. Works wonders for difficult cases
> such as inserting 15 aa (the longest I've tried).
Good idea, I'll try that with something that I'm having problem with
at the moment (the oligos were designed wrongly so may not anneal
> Oh, and if anyone is interested: QuikSolution Reagent is
> unadulterated pure DMSO. In other words, buying PfuUltra
> from Stratagen, DpnI fron NEN and having decent home made
> electro-competent cells is equivalent or better to the useful
> kit content in QuikChange II.
What transformation efficiency do you get with your electro-competent
cells? I inherited an electroporator from a previous occupant of the
lab, and so far I haven't been able to do better than my chemical
competent cells (~1-3 x 10^8 cfu, using Inoue method). It may be the
electroporator (it is a bit flakey), but wondered why I can't get to
10^9-10^10 cfu which is what is claimed possible.
> Speaking of QuikChange:
> I noted Stratagene now sells QuikChange® Multi Site-Directed Kit.
> Judging from description, it contains a mix of polymerase and ligase.
> The idea is, basically, to synthesize enough single stranded circular
> DNA and let E.coli take care of 2nd strand synthesis. This is
> accomplished by using a mix of polymerase and ligase. (I think
> the idea was triggered by observations that i) just one primer works
> sorta OK in otherwise normal protocol, ii) using two pairs of primers
> to make two mutations simultaneously works too, albeit with
> lower efficiency, ~ 30% in our lab).
> Of course, Stratagene does not really tell what ligase they add
> and what substrate it takes - which allows them to raise the price
> of the kit by 1.5X. Anyone knows of any commercially available
> thermostable DNA ligase?
Don't know, but you can probably clone it out yourself if you are
going to use quite a lot of it. I think I may have a little
chromosomal DNA from Methanococcus jannaschii left over from some
years ago, so might take a look one day.
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