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 <[email protected]> > > > > > 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 > > [email protected] > > http://www.bio.net/biomail/listinfo/methods > > > _______________________________________________ > Methods mailing list > [email protected] > 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) _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
