Help with DIG Northerns

R. Rex Denton DENTON at biomed.med.yale.edu
Mon Apr 29 14:51:21 EST 1996


In <4m16e6$340 at hobyah.cc.uq.oz.au> Helen writes:

> I am doing DIG Northerns using the total Boehringer system, but 
> am  having trouble stripping and reprobing the membranes.  The 
> membranes are Boehringer membrane, RNA is UV fixed after 
> transfer.  I am using DIG labelled RNA probes, with DIG Easy 
> Hyb hybridisation fluid.  All reagents used for stringency wash 
> and DIG detection are made up RNase free. > The first hyb with probes for low expression level targets 
> works well. A second hyb, even for loading control such as GAPDH, has 
> never been successful in my hands.  Staining of the membrane 
> with methylene blue at various stages seems to indicate that 
> either the RNA is being degraded during washes and detection, 
> or is being masked.  No one step seems to be the problem.
> 
> Do you have any suggestions for solution?
> 

Dear Helen,
I've been where you are now presently.  I traced the problem to inadequate
fixation of the RNA to the membrane.  As a consequence, the target is stripped
off the membrane with the probe during the stripping step.  I also found that
there was a dramatic difference in the type of membrane that was used for the
blotting.  The best one in my hands was the was the Boehringer membrane 
(no affiliation, blah,blah) that they pitch with the kit. (I ttried about 4
different kinds)

The best fixation that I acheived was to dry the blot
at 60 C for 1h then cross-link using
the germicidal lamp in a Baker biogard tissue culture hood for exactly 2 min.
I make no guarantees but I'm rather sure that this is your problem.  This is
optimal for the Boehringer membrane.

By the way, If you find a good membrane, let me know about it.  These
manufacturers have been playing switching games for the past 4 yrs or so, and
what works today may not work as well the next time that you order the same
product!!!!





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