fluorescence PCR

[Deborah B. Loeb]

In article <4emqj6$7fg at damage.usa1.net>, Sui Huang
> Children's Hospital, Boston (suihuang at usa1.com) wrote:

> Does anybody has experience with visualizing PCR products 
> (in agarose or PA gel) using 5' fluorescence labelled primers 
> (Amersham) and the Fluorimager (Molecular dynamics) ?
> Especially, how sensitive is this method compared to 
> 32P-nucleotide incorporation ?

Yes! it works. My erstwhile lab at Duke U. bought a Mol.Dy. fluorimager
for the purpose of reading PCRs (PAGE) without radioactivity. It is seems
to be only a little less sensitive than 32-P incorp., but then, 5'
end-labeling would be.

Since we design many of our own primers, it is more cost-effective to use
the SYBR green fluorescent dye to stain (PA and agarose) gels after
running them. We found that the method is less sensitive but cleaner (re:
background), and the image can be modified by the software. A band
analysis and an Excel-type program can be used to read data automatically
into a table.