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
