PCR failing

Irit Rappley via methods%40net.bio.net (by irappley from scripps.edu)
Fri Apr 9 11:18:23 EST 2010


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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