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


Hi,

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 
crosslinking?
And when is it bad to dry it? Is it necessary to moist the membrane before 
prehybridisation?
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 
problem.
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,
Simone 




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