"reverse northern" problems

eheinecke at STUDENTS.WISC.EDU eheinecke at STUDENTS.WISC.EDU
Thu Dec 5 13:08:20 EST 1996


I am currently working with mRNA differential display.
Having cut out and subcloned several possible differentially 
displayed bands, I am attempting to confirm differential expression
using the "reverse northern" technique.  I realize Northerns are the
ultimate confirmation method, but I'm trying to develop this dot 
blot technique as a quick screening method for candidate cDNAs.
To make a long story short, I've tried dot blotting both mini-preps of
my subcloned cDNAs and PCR-amplified cDNAs, followed by probing
with 32P-labeled (random primed) cDNAs from 2 separate strains of the 
organism we work with. Both the control for my probe and another
control cDNA from the organism are showing up.  Unfortunately, I
have yet to see anything hybridizing to the cDNAs of interest.
I may try longer hyb. times, since these may be rare mRNAs. 
Any other suggestions?

Liz Heinecke
U.W. Madison



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