EB assay for DNA (was InVitrogen Dipsticks)
Bill Melchior, NCTR/FDA
wmelchior at NTBTOX.NCTR.FDA.GOV
Thu Aug 20 09:14:26 EST 1992
>If working with Ethidium Bromide holds no fear for
>you, you might try a fluorescence spot test we often use. Briefly, 2 ul
>droplet of the DNA whose concentration is spotted on saran wrap layed on a
>UV transilluminator. An 8 ul droplet of a .5 ug/ml solution of EtBr is mixed
>by pipetting up-and-down. A series of droplets containing known quantities
>of DNA are also spotted. The fluorescence of the test sample is then compared
>to the knowns. Like the Dipstick, the best response is obtained with amounts
>as low as 1 ng an as high as 10 ng. A picture can taken with the appropriate
>filter for a permanent record (and also to avoid having to stare at a UV box).
>When quantitating, be sure to include a zero ng control, since the EtBr itself
>can fluoresce. Also, ignore the fluorescence seen at the edges of the sample.
>Once the solutions of EtBr and the known samples are prepared, this method
>takes only a few minutes to do.
I use essentially the same method. I have a microscope slide, the surface
of which I treated with silicone grease (which practically never comes off)
to keep the drops from spreading. After running the test, I just wipe off
the droplets and stick the slide back in the drawer.
I've not made a careful study, but feel that I get the best results, over
a wider range of DNA amounts, when the WEIGHT of the added ethidium bromide
is roughly equal to to the WEIGHT of the DNA. As stated above, it's crucial
to have a blank spot in the series.
The opinions stated are mine, not those of NCTR or its sponsoring organizations.
Bill Melchior || "You have lawyers the way
National Center for Toxicological Research || other people have mice."
Jefferson, AR 72079 ||
(501) 543-7206 || -Kenneth Duncan, English
|| Health & Safety Executive,
WMELCHIOR at NTDOC.NCTR.FDA.GOV || to US regulators
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