PCR failing

LeboMad via methods%40net.bio.net (by lmadubanya from gmail.com)
Thu Apr 15 03:45:48 EST 2010


On Apr 9, 6:18 pm, Irit Rappley <irapp... from scripps.edu> 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
> > _______________________________________________
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> > Meth... from net.bio.net
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>
> - 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


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