long storage of northern blot

GWDGV1::KCHOWDH kchowdh at gwdgv1.dnet.gwdg.de
Wed Nov 24 08:28:24 EST 1993


Hi Christian,
The hyb. condition is : 50% formamide, 1M NaCl, 1% SDS, 100ug/ml denat. SS DNA,
42 degrees C. Washing is with 1% SDS with different SSC conc. as needed.
I think the success of northern depends on putting large amount of high
quality RNA on the blot to start with. Once you have this you can reprobe it
many times.  Of course with every reprobing you will loose little bit of the
signal intensity.  But for most cases it will not be a big problem. I have
not compared other positively charged membranes and still doing all northerns
on gene screen plus membrane.
One thing I found that significantly reduces the background is to prewash
the blot before prehybridization with 2XSSC-1%SDS at 65 degrees C for 1 hour.
This removes all contaminants that make the probe stick to the filter.
cheers,
Kamal.



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