pcr prob

Aawara Chowdhury via methods%40net.bio.net (by aawara from pontiff-playground.org)
Sun Feb 24 15:46:21 EST 2008


In <1203860754.14229.0 from proxy00.news.clara.net>,
 ChenHA <hzhen from freeuk.com> wrote:

> harold b wrote:
>> Hi, my negative control (no template with both primers and mastermix)
>> consistently shows a stronger single band whereas my amplicons show a
>> leading similar but weaker band followed by a fading smear reminiscent
>> of non-amplified DNA. In each case the product with DNA is less
>> intense. Can someone make sense of this?
>
> The strong band is most likely just your primers.  Easy to check by 
> running the same amount of primers used on the gel.  

The strong band is unlikely to be "just your primers".  As someone 
earlier in this thread described, the strong band is what has 
quixotically been labeled as "primer dimer" - the result of DNA synthesis 
after a primer anneals (partially) to itself or the other primer in the PCR.

Loading 5 or 10 picomoles of each primer on an agarose gel by itself
will not give a band that fluoresces brightly in the presence of 
ethidium bromide.

AC
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