kmorris1 at opal.tufts.edu
Tue Apr 30 17:27:17 EST 1996
In article <4m2imt$i8o at newsbf02.news.aol.com>, griffcouch at aol.com (GriffCouch) writes:
> Does anyone have a protocol for unblotting? I understand that it involves
> Northern hybridization to a dried out RNA gel as opposed to transfer and
> hybridizing to nylon.
I've used an unblot protocol for Southern analysis with oligo probes, but never
for Northerns. Basically, I denatured and neutralized the gel as for a regular
Southern, then put it on two sheets of Whatman paper and dried it on a gel
dryer until flat. DON'T use a vacuum blotter, it's far too strong. I hooked
it up to house vacuum and it generally took about an hour or so to become flat.
Unlike polyacrylamide, you can peek to see if it's done. After it's flat, turn
on the heat to about 60 degrees C for about 20-30 minutes, then turn off. The
gel should look like a clear piece of "fruit leather", not like cellophane.
Then you can go ahead and hyb. Since my protocol was for oligos, they won't be
useful here, but email if you're curious of if you have other questions.
Kelly Thome PhD kmorris1 at opal.tufts.edu
Brigham and Women's Hospital
Boston, MA <standard disclaimer>
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