hybridisation / stringency
kawalekm at earthlink.net
Mon Oct 4 20:38:16 EST 2004
It's the relationship between the salt concentration and the repelling effect of the negatively
charged DNA backbone. The Na+ ions bind to the PO4- groups on the DNA neutralizing their charge.
As the charge disappears, the hydrophobic interactions of the base is greater than the repelling
force, so the DNA forms double strands. During the hybridization the salt concentration is about
0.9 Molar. That is much higher than physiological concentration and strongly favors double
strandedness. As you drop the salt concentration in the washes, the backbones becomes progressively
more negatively charged and this forcex the strands apart.
On 11 Jun 2004 03:48:40 -0700, alion85 at hotmail.com (redeamer) wrote:
>"jonner" <jonner at bigfootREMOVE.com> wrote in message news:<caab5m$s8o$1 at dux.dundee.ac.uk>...
>> 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,
>Man, check this out:
>---> Cheers, Drew
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