Help with DIG Northerns

Gary Moore Gary_Moore-1 at sbphrd.com
Tue Apr 30 10:52:19 EST 1996


Helen Haase <h.haase at mailbox.uq.oz.au> wrote:
>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.   I use Boehringer 
>blocking solution in detection step. Hybridisation bottles in a 
>Hyb oven is used for Hyb, plastic trays on a shaking platform 
>used for  stringency wash and DIG detection step.
>
>The first hyb with probes for low expression level targets 
>works well.  Stripping has been attempted using a number of 
>methods, but usually by placing the membrane in boiled 0.1% 
>SDS.  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?
>
>Thank you, Helen
>
>

We also encountered this problem when we set the procedure up in our lab.
The reason why we couldn't successfully reprobe was probably due to the 
membrane being stored for 1 or more days soaked in the chemiluminescent 
substrate after exposing to film (prolonged exposure to the high pH of 
the substrate buffer was perhaps causing degradation of the blotted RNA).
We overcame the problem by stripping the membrane immediately after the 
exposures were completed and either sealing it wet (soaked in 2XSSC) in a 
clean polythene bag in the fridge or prehybridising it prior to 
reprobing. The stripping procedure we use is as follows:

DEPC-water for 5 min
Boiled 0.1% SDS for 10 min
100 mM maleic acid, 150 mM NaCl, 0.3% Tween 20, pH 7.5 (i.e. Boehringer
1X Washing buffer) for 5 min
2X SSC for 5 min
Repeat last step.

(All steps performed on a rocking platform.)



Hope this is useful.


Gary Moore
 
-- 
The opinions expressed in this communication are my own,
  and do not necessarily reflect those of my employer.





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