semi-dry blotting of dna
chihara at usfca.edu
Mon Aug 18 00:41:23 EST 1997
John Herbst wrote:
> We've been trying to transfer DNA from agarose gels (1%) in 1xTBE
> to Nytran nylon membranes, however, we've only met with partial success.
> The problem seems to be buffer breakdown-possibly from the Pt/Ti anode-
> and subsequent gas release and bubble formation; end result is
> inconsistent DNA transfer due to gaps forming between gel and membrane.
> The gel has gone through typical treatment for Southerns before
> transfer, i.e. NaOH denature; neutralize in Tris,HCl; and equilibration
> in 1xTBE-this is recommended transfer buffer as communicated by Fisher
> Biotech staff. I've heard that methanol is used in Western blots to
> increase the resistance of proteins, thus making them stick better to
> the membrane. My question is this: are there any reagents which can be
> added to the transfer buffer to inhibit breakdown and gas formation, or
> which will make the DNA stick to nytran better and thus allowing less
> current to be used?
> NOTE: We are using 50 mA, 10 Volts as settings on our power supply (0.5W)
> and take between 2-3 hrs to transfer, as Fisher Biotech recommends.
> I'd greatly appreciate any suggestions or info on reagents or
> parameters which others have found to be successful in improving this
> process in their labs, or knowledge obtained from others experiences
> with this technique.
> John Herbst
Are you sure the bubbles are forming and not already between membrane
and gel? We found that rolling a pipet like a rolling pin over each
layer as it goes down really helped. Also add a little extra buffer
on the top then roll again. Hope this helps.
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