PCR failing

Peter Ellis via methods%40net.bio.net (by pjie2 from cam.ac.uk)
Fri Apr 9 05:20:55 EST 2010

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.


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