semi-dry blotting of dna

John Herbst jmherbst at FACSTAFF.WISC.EDU
Mon Aug 18 19:17:37 EST 1997


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.
Thanks! 
John Herbst



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