Site-directed mutagenesis problem

Rosario Díaz - González via methods%40net.bio.net (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,
easy to download from several lab websites.

Rose.

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...
> 
> Sebastien
> 
> 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
> > _______________________________________________
> > Methods mailing list
> > Methods from net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www.bio.net/biomail/listinfo/methods


Rosario Díaz-González, PhD
Instituto de Parasitologia y Biomedicina "Lopez-Neyra"
Avda. Conocimiento S/N
Parque Tecnologico Ciencias de la Salud
18100 Armilla - GRANADA
Spain
Tlf: 958181621
Fax: 958181632
http://www.ipb.csic.es
--
Open WebMail Project (http://openwebmail.org)



More information about the Methods mailing list