The set of primers that gives you multiple bands is non-specifically binding to other areas if your Sna sample. You could first try to increase the annealing temp (temp at which the primers bind to the template) and see if that helps. If that doesn't work you can try redesigning your primers a little longer (usual length us 20-22 bp). Both of the above factors will increase specificity and reduce smear or multiple bands.
I would also try to go in genome browser website and try to blast then to the genome and see if they are binding to multiple sites. We used to always do that before trying any PCR or ordering primers. Good luck! Sent from my iPhone > On Mar 12, 2014, at 11:04 AM, June May <[email protected]> wrote: > > Hi all, I have a PCR problem and I can really use some help. This is an > established protocol we've been using for genotyping for a while. I'd say > half of the time it works beautifully. Sometimes we get multiple bands. > Sometimes I get smears for one set of primers but not the other set. Once I > got nice bands for some DNA sample but smear for others while I extracted > those samples together. I use the DNA sample for other PCRs and they work > just fine. I use my mastermix and PCR machine for other PCRs too so I don't > think there's a problem there. Can anybody give me some pointers as to why > this keeps happening? > > Thank you very much! > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
