Dear Eva, I think your problem is either the annealing temperature, in our lab we usually use something between 48 and 52 °C. Or your elongation time is too short. Depending on the polymerase used, you should give it about 2 mins for every 1000 bp of your vector+insert. Good luck! Niko
> ------------------------------ > > Message: 8 > Date: Tue, 18 Jan 2011 13:16:12 +0100 > From: Pepa Florez P?rez <[email protected]> > Subject: Site-directed mutagenesis problem > To: <[email protected]> > Message-ID: <[email protected]> > Content-Type: text/plain; charset="iso-8859-1" > > > 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 This message and any attachment are intended solely for the addressee and may contain confidential information. If you have received this message in error, please send it back to me, and immediately delete it. Please do not use, copy or disclose the information contained in this message or in any attachment. Any views or opinions expressed by the author of this email do not necessarily reflect the views of the University of Nottingham. This message has been checked for viruses but the contents of an attachment may still contain software viruses which could damage your computer system: you are advised to perform your own checks. Email communications with the University of Nottingham may be monitored as permitted by UK legislation. _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
