Site-directed mutagenesis problem
Rosario Díaz - González
(by rdiazg from ipb.csic.es)
Tue Jan 18 12:48:39 EST 2011
You also might find help in the QuickChange pdf User's Manual of Stratagene,
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On Tue, 18 Jan 2011 07:57:44 -0500, Sébastien Vigneau wrote
> Maybe you have a lot of non-specific amplification. Does your template/oligo
> sequence contains repeats? Or could your oligos possibly anneal somewhere
> else on the template? Increasing the annealing temperature of the PCR
> reaction could also help...
> Maybe something in the reaction mix (e.g. the enzyme) gets stuck to this
> specific sequence. You could check that by doing a phenol/chloroform
> purification before to migrate on a gel...
> 2011/1/18 Pepa Florez Pérez <snipeurope from hotmail.com>
> > Dear all,
> > I am writing to you becasue I am experiencing problems to carry out a
> > site-directed mutagenesis.
> > I use two oligonucleotides 22 nts. long, that hybridize completely but not
> > on a single-base located in the middle of the sequence.
> > What I get after carrying out the PCR is a smear in the gel:
> > - The enzyme is not a problem since the positive control (another plasmid
> > same lenght with different oligos did work)
> > - I get a smear no matter which vector I use as template, if I use the new
> > oligos, but not the others.
> > - If I increase template concentration I get the same result.
> > Can you guess what it is going on? What else would you try? I am really
> > lost.
> > If any of you is interested I can send you a summary picture too.
> > Thank you very much
> > Eva
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Rosario Díaz-González, PhD
Instituto de Parasitologia y Biomedicina "Lopez-Neyra"
Avda. Conocimiento S/N
Parque Tecnologico Ciencias de la Salud
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