ribo- or RPL probe ?
Malcolm Moos Jr.
moos at helix.nih.gov
Sat Nov 5 14:10:38 EST 1994
> I am experiencing extreme problems with my northerns and southerns (both
> genomic). Basically, I label my probes with RPL, like the Amersham
> kit. However, even though I get quite hot probes (10E7 cpm) I see only
> things like rRNA lighting up.
> So, I was thinking that this could be due to too small DNA fragments
> after labelling (I didn't test this on a gel).
> So, now I want to try making a riboprobe, a liitle bit more work, but
> Anybody out there who knows how to compare both these labeling
> systems ? Is a riboprobe hotter and better ?
10E7 is not anywhere near hot enough. You should get 2x10e9/µg with random priming. Try reducing the amount of probe you put into the random priming reaction. You might also try downward alkaline capillary blotting, which is not only easier, cheaper, and faster than other methods, but is said to give better sensitivity.>
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