hybridisation / stringency

redeamer alion85 at hotmail.com
Fri Jun 11 05:48:40 EST 2004


"jonner" <jonner at bigfootREMOVE.com> wrote in message news:<caab5m$s8o$1 at dux.dundee.ac.uk>...
> Hi,
> 
> After looking through the archives, I haven't been able to find an answer
> for this.
> 
> 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.
> Many thanks,
> 
> jonner.


Man, check this out:

http://dicty.cmb.nwu.edu/Chis_lab/Lab%20Manual/molecular_biology.htm

---> Cheers, Drew



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