Help with UV Shadowing requested

[David Micklem]

In article <46lt28$n57 at taco.cc.ncsu.edu>, smlane at unity.ncsu.edu (Stephen
Marcus Lane) wrote:

>Hi, I'm a grad student in Biochemical Engineering here 
>at NCSU, and I'm having real trouble getting a
>UV shadowing protocol to work.  I haven't been able
>to find much at all in the literature or protocol
>manuals on the technique.
>
>UV shadowing is supposed to work by running RNA or SS DNA
>on an acrylamide/urea gel, then exposing the saran-wrapped
>gel to UV light.  The light is absorbed and fluoresced
>by a TLC plate (I'm using Brinkmann: Merck silica gel
>F254), except where the RNA/DNA is on the gel.  In these
>spots the RNA/DNA absorbs the flouresced light, supposedly
>showing up as "shadows" on a green background.
>
<snip>
>
>Thanks, Steve
>--
Hi Steve,

One other thing (than the only other reply I've seen...) - 

Are you getting the geometry of your set up right?!  It needs to go:

UV source
GEl on saran wrap
TLC plate (or other fluor)

ie, your gel must be between the UV source and the fluor - the RNA/DNA is
NOT absorbing the fluoresced light, it is absorbing the UV and preventing
it exciting the fluor.  Hope this helps,

David

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D.R.Micklem,
Wellcome/CRC Institute,       Time flies like an arrow...
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Tel: [+44] (0)1223 334129     Email:drm21 at mole.bio.cam.ac.uk
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