(by aawara from pontiff-playground.org)
Mon Feb 25 07:32:54 EST 2008
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
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
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