Problems with QuickChange mutagenesis kit

Yulia Shifrin yulia24 at gmail.com
Fri Jun 9 09:35:56 EST 2006


>
> Try adding 10% DMSO to the PCR reaction mixture. Worked great for me for
> fragments as large as 15kb. Good luck!
>
>
> On 6/9/06, María Jesús García <mjgarcia at cnb.uam.es> wrote:
> >
> > Hi everybody!
> > I´m looking for some help with the QuickChange mutagenesis kit from
> > Stratagene.
> > I want to make a single aminoacid change in a
> > protein (1.1kb) cloned in pcDNA3.1. (total lenght
> > of the construct 6.5kb). I tryed with 50 and 75ng
> > of DNA template and 125-150ng of primer. I´ve
> > design the primers according to the manufacturer
> > and I've used the following conditions for the
> > PCR:
> > Segment 1:
> > 1 cycle 95ºC 30 seconds
> > Segment  2:
> > 16 cycles 95ºC 30 seconds
> > 55ºC 1 minute
> > 68ºC 13 minutes (2 minutes per Kb)
> > The problem is that after transformation I DON`T GET ANY COLONIES AT
> > ALL.
> > I´ve used also the control provided with the kit
> > and I don´t get any colonies at all as well.
> > So, could somebody advise me on where I might be going wrong?
> > Thank you.
> >
> > Maria. J. Garcia
> >
> >
> >
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> > Methods at net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
>
>


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