Hello Please use internet primer3, primer check and blast. Use 5 degrees more in annealing temperature and 1.5 mm to mM of MgCl2 for PCR mixture. If u still have problems change primers again or polymerase. Good luck
Envoyé de mon iPhone Le 22 mars 2014 à 01:40, "Silky" <[email protected]> a écrit : > 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 _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
