Is downward transfer REALLY good for Northern?

Jason Lei 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.


Jason Lei
-- 
				   Jason Lei                            
			         Horticulture				
			       Purdue University                    
			    West Lafayette, IN 47907             



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