Is downward transfer REALLY good for Northern?
leij at sage.cc.purdue.edu
Thu Aug 25 00:44:18 EST 1994
I was trying to do Northern by using alkaline (0.05 N NaOH) cross linking
(see Biotechniques, June 1994) since it was claimed that this method can
increase the detecting sensitivity for rare messages. Essentially, the
formaldehyde gel (with EtBr in the sample) was treated with 1% glycine for
30 min, 0.05 N NaOH for 20 min, and 20X SSC for 40 min before transfer.
Although the authors did transfer (upward?) overnight, I decided to do
downward transfer by using 20X SSC as the transfer buffer because I already
had had good results for Southern with downward (10X SSC). However, to my
surprise, the RNA was still in the gel after transfer although I am not sure
if any RNA had been transferred. Before trying this, I had been doing
overnight upward transfer with 20X SSC for Northern without problem. One
neighbor told me he also had problem doing downward for Northern (alkaline
transfer in his case) while regular upward worked well. He also had good
luck with downward for Southern. Oh, almost forgot, the membrane I used
was Nitran Plus, the gel was about 7-8 mm in thickness, and transfer took
1 h 45 min.
I am aware of some papers about downward for both Southern and Northern, but
did we (me and my neighbor) miss anything in our trying? I also know some
people do not treat their RNA gels before transfer, but I don't think the
pre-transfer treatment would cause the problem.
Any comment appreciated.
West Lafayette, IN 47907
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