Site-directed mutagenesis troubleshooting

Dave Morgan d.d.morgan at ncl.ac.uk
Mon May 10 07:14:49 EST 2004


I initially had trouble with this kit when trying to mutate a 12kb plasmid.
I found that including "normal sequence" primers (with 5' phosphate) that
anneal to the plasmid every 1.5 to 2kb helped enormously and gave me loads
of mutated clones.


"Wolfgang Schechinger" <hubahopp at gmx.de> wrote in message
news:3.0.6.32.20040510114322.01465950 at pop.gmx.net...
> Dear ?
>
> increase the number of cycles to 25 and extend for up to 10 min. if this
> does not work, decrease the annealing temp. when you run a sample (before
> the DpnI) in agarose, can you see anything?
>
> Wo
>
>
> At 07:54 10.05.2004 -0000, stoychevs at biology.biol.wits.ac.za wrote:
> >I am attempting to create a truncated mutant by introducing a stop codon
> into my primers using Stratagene's Quickchange Site-directed mutagenesis
> kit. I am experiencing problems with my PCR since after transformation I
> don't get any colonies on the plates (100mg/ml Amp). I know that the
> transformaiton works since I transformed the wild-type plasmid
> successesfully and got 100s of colonies. I have tried varying my template
> DNA concentration (0.7ng - 100ng) while keeping the primer concentration
in
> excess (600ng) with no success. My plasmid (pGEX-4t-1) together with the
> insert is 5.6kb. I use the following conditions for the PCR:
> >Segment 1: 1 cycle at 95C for 30sec
> >Segment 2: 16 cycles 95C for 30sec
> >55C for 1min
> >68C for 6min
> >Could you please advise on where I might be going wrong. Thank you.
> >
> >
>
>http://biowww.net/mynews/tree.php?group_name=bionet_molbio_methds-reagnts&b
> egin=0
> >
> >
> Wolfgang Schechinger
>
>
> ---





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