hybridisation / stringency
jonner at bigfootREMOVE.com
Thu Jun 10 13:59:10 EST 2004
After looking through the archives, I haven't been able to find an answer
In a Southern hyb, the recommended protocol for a commercially-available
"rapid" hyb buffer calls for hyb'ing at 42oC (the buffer contains 50%
formamide). OK, fine.
Now, for the washes, the "high stringency" wash is 0.1X SSC / 0.1% SDS at
42oC. I'm assuming that any formamide present in the hybridisation has been
washed away by this point, so why aren't these washes performed at the
standard conditions of about 60oC? Is there something I'm missing, or is my
above assumption not correct? To be honest, in reality it doesn't seem to
matter, since in the 3 years or so that I've been using this hyb buffer,
it's given fantastic results without the appearance of non-specific bands.
So, I guess what I'm asking is: *why* does this work? Is the experiment's
stringency controlled more by that of the initial hyb'ing and less by the
washes? I would love to get this clear in my head, but have failed to find
any useful information.
If anyone can shed any light on this, I would be extremely grateful. It's
really doing my head in.
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