pcr prob

Aawara Chowdhury via methods%40net.bio.net (by aawara from pontiff-playground.org)
Mon Feb 25 07:17:26 EST 2008


In <1203893436.24100.0 from proxy01.news.clara.net>,
 ChenHA <hzhen from freeuk.com> wrote:

> How do you know how much primers the original poster added?  

The amount doesn't matter - see below.

> It is for 
> the original poster to comfirm or deny, or check by runnning the gel (as 
> I stated, by using the same amount), not for you to assert.  Whoever 
> gives you the idea the 5 or 10 picomoles is what everyone uses?  I 
> don't, and I have done perhaps thousands of successful PCR reactions. 
> And they do fluoresces, how brightly of course depends on how much you add.
>
> As it happens, I see this kind of things quite often, from my own PCR.

Ethidium bromide binds single-stranded DNA extremely poorly, if at all.
It fluoresces when bound to RNA, simply because most RNA molecules do
fold-back upon themselves to form partial duplexes.  Long ssDNAs also
fluoresce in the presence of ethidium for the same reason.

I find it highly improbably that your ss oligonucleotides snap back on
themselves sufficiently to fluoresce with ethidium, in the absence of
polymerization that makes them ds.

AC
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