pcr prob

[Aawara Chowdhury]

In <qlywj.9872$0M3.986 from newsfe17.lga>,
 Aawara Chowdhury <aawara from pontiff-playground.org> wrote:

> 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 improbable that your ss oligonucleotides snap back on
> themselves sufficiently to fluoresce with ethidium, in the absence of
> polymerization that makes them ds.

BTW, there are several good papers on why ethidium fluoresces when bound
to double-stranded nucleic acids, but not single-stranded ones.  Here
are two:

Decay of Fluorescence Emission Anisotropy of the Ethidium Bromide-DNA
Complex Evidence for an Internal Motion in DNA.  Wahl, Paoletti, & Le Pecq
PNAS 65:417-421 (1970).

Ethidium Bromide Does Not Fluoresce when Intercalated Adjacent to 
7-Deazaguanine in Duplex DNA.  Latimer & Lee. JBC 266:13849-51 (1991).

The latter has several citation which examine in some depth the 
mechanisms by which ethidium bromide fluoresces when bound to nucleic
acids.

AC
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