On Apr 9, 6:18 pm, Irit Rappley <[email protected]> wrote: > You could also try getting the 260/280 reading for your DNA in a > spectrophotometer to make sure it's relatively pure (you want a ratio > of 1.8-2.0). While you're at it, use the 260 reading to check the > concentration of your DNA prep. > > Also, when you say that you've done all the troubleshooting, do you > mean that *you* have personally done it with your own hands, or that > your student has done it? I had a similar situation with a PCR that > suddenly stopped working for a technician, and in the end I had to > take over the troubleshooting myself to figure it out. > > Irit > > On Apr 9, 2010, at 3:20 AM, Peter Ellis wrote: > > > > > On Fri, 9 Apr 2010, LeboMad wrote: > > >> Hey Duncan thanks for the response. You're right it's 0.2mM it was > >> just a typing error. The problem is not really the multiplexing > >> because it worked in the past perfectly I could attach a gel or > >> two if > >> I could. > > > OK, so something's changed. What? New batch of primers, new batch > > of DNA > > extraction reagents, new batch of enzyme / buffer / NTPs? > > > I would try the following: > > > 1) Positive control PCR with a known good primer pair + template, to > > confirm that the enzyme / buffers / nucleotides are OK. This needs > > to use > > a primer pair that's not part of the multiplex reaction, since you > > don't > > know if those primers are working OK. > > > If the enzyme / buffers / nucleotides check out, then: > > > 2) Test each of the primer pairs from the multiplex individually > > against a > > known good template (i.e. a DNA prep that's previously given a good > > band). > > This tells you if any of the individual primers has degraded or become > > contaminated with a PCR inhibitor. > > > If the primer pairs from the multiplex are working OK, then the > > problem > > may lie in your DNA samples, so: > > > 3) Test the failing DNA samples with some other primer pair that > > should > > always give a band (e.g. beta actin for mammalian samples). This > > tells > > you if something is going wrong with the DNA preps. > > > If you work through these stages in turn, that will confirm the > > integrity > > of your PCR reagents, the primers and the template. Hopefully this > > will > > pinpoint the problem. > > > Peter > > _______________________________________________ > > Methods mailing list > > [email protected] > >http://www.bio.net/biomail/listinfo/methods- Hide quoted text - > > - Show quoted text -
Thanks to everyone for commenting and providing me with tips to troubleshoot. I took the advice of separating the primers as they tag separate QTLs. It's now working and no dimers. I need the 3 QTLs in the progeny. One QTL is missing hence the marker is absent. interestingly when I mutiplex with only the two that work, I get results. The third marker just complicates things. I had got to a point where I mixed 2 ul of each PCR product to see the presence of the 3 bands in each well. But since the multiplex is working with the two markers I am happy. Thanks again guys Kind regards Lebo-RSA _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
