pcr prob

Aawara Chowdhury via methods%40net.bio.net (by aawara from pontiff-playground.org)
Mon Feb 25 09:25:25 EST 2008


In <TXzwj.101$Td5.25 from newsfe07.lga>,
 DK <dk from no.email.thankstospam.net> wrote:

> FWIW, every time my PCR did not work or produced low yield, 
> a diffuse and very weakly stained band below 100 bp can be seen. 
> I always interpreted it as "unused primers". Granted, typically the 
> amount loaded on the gel would correspond to < 50 ng and it is
> doubtful that EthBr has this sensitivity for ss DNA. Still, the fact 
> is that whatever the primers do (dimers, hairpins) they do 
> bind EthBr. 

Dima - agreed completely.  But your PCRs contain polymerase and dNTPs;
so your "unused primers", are in fact the primer-dimer products that
are the result of primers annealing to themselves, and permitting
the synthesis of dsDNA (that binds ethidium) in the presence of dNTPs
and polymerase.

Try doing this same reaction without polymerase, you will not see
the fluorescent band (or without dNTPs for that matter).

Ethidium requires base-stacking to fluoresce.  It doesn't intercalate
into adjacent base-pairs because of steric hindrance.  And a minimum
of three stacked ethidium molecules are needed for fluorescence to
be observed.  Thus the minimal ds nucleic acid that does fluoresce
with ethidium is 6 bp in length (work published by someone who used
to be UW-Madison - Khorana).

Therefore, for oligonucletides to fluoresce directly with ethidium, 
they must form at least contiguous base-pairs, a scenario that is 
certainly possible, but high unlikely for PCR primers.

AC
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