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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