handling with northern blots/membranes

Simone Marker via methods%40net.bio.net (by marker from rhrk.uni-kl.de)
Thu Sep 13 03:46:37 EST 2007


I'm doing my first northern blot and I have found so many different receipts 
and handling methods that I am finally more unconfident than before...
My questions are:
When is it necessary to dry the membrane? Do I have to dry it before 
And when is it bad to dry it? Is it necessary to moist the membrane before 
For a second hybridisation I heard that the membrane should not be dried 
after hybridisation but immediately stripped. Is that true or is it possible 
to strip a membrane that was dry before? I used 0,5% SDS (100°C, 10min) to 
strip a membrane that was stored dry for two days. The removal of the probe 
was not complete, but I'm not sure if the drying of the membrane was the 
Another point:
Can I reuse a hybridisation solution with probe in it (DNA probe) and if 
yes, how often? (I use 33P radiolabeled probes, I have to consider the 
halflife of P33, but how stable is a DNA probe in hybridisation in general?) 
And what about denaturation of the probe when I reuse the hybridisation 
solution? Is the probe stably singlestranded or do I have to boil the 
solution before use?

I appreciate any practical hint, thank you in advance,

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