geha at hsc.usc.edu
Wed Nov 12 13:52:47 EST 1997
In article <64ci66$8fk_001 at leeds.ac.uk>, BMBTJY at leeds.ac.uk (T.J. Young) wrote:
> Dear All,
> I was wondering if anyone has experienced any problems with the stratagene
> QuickChange kit. The first time I used the kit it worked an absolute treat
> with the control Whitescript reaction giving >200 colonies which were near
> enough all blue. However, the last couple of times I've used the kit I've
> got nothing. That is to say, one or two blue colonies with the control
> reaction and none with my desired mutated construct. The kit is now about
> 3 months old. The competent cells are still good giving >10e8 cfu. I've
> checked the Pfu and dNTPs with a PCR of about 800bp that I know works and
> they seem fine too so I am completely stumped. I've talked to Stratagene
> and they can't understand it either. I know that you can play around with
> your template concentration to give greater efficiency but that's not the
> problem because the control reaction isn't working either. Any ideas would
> be greatly received.
> Thanks in advance,
> Tim ;-)
Tim, I've had the exact same problem and the company wasn't of much help either.
But after doing a few runs, the problem was easily fixed.
Do not worry about your template concentration, it is not the problem and
neither is the Pfu, the dNTPs or the competent cells. The problem is
the annealing temperature. The recommended 55 degrees is simply too high,
especially for mismatched oligos since your doing mutagenesis. Simply lower the
annealing temperture from 55 to 52. It works great with me.
I hope this helps.
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