How to check if DNA is transfered
hsibul at ebc.ee
Thu Mar 25 05:22:53 EST 1993
Charles Rivers (RIVERS at IPRSH.DOM.UAB.EDU) wrote:
: Does anyone know of a way to check and see if any DNA has transfered to a
: nylon membrane after a neutral, capillary southern blot? I know that you
: can check the collapsed gel, but what about the membrane? Here's the
: problem: I tried to transfer 5 micrograms of digested DNA in a 0.75% agarose
: gel. I noticed that my gel had collapsed VERY early--like within an hour;
: this made me very nervous, but I let the transfer continue. When the transfer
: was complete (from time), I restained the gel to see if the transfer was
: indeed complete--it wasn't. As a matter of fact, it looked like nothing
: transfered at all. Being spring and all, I've gotten lazy and don't want
: to repeat an entire digest series. I just want to be able to check the
: membrane and see if any of the DNA transfered. I seem to recall that there
: is a way of staining the nylon membranes with methylene blue to see if any
: DNA is bound, rather than wasting time with a legnthy (sp?) hybridization
: and detection. Any information would be very appreciated.
Comparing the intensity of the etbr glow in UV before and after
the transfer is not such a bad method after all.
Also, if you check the intensity of the membrane, it should give you
some idea of whether the glorious transfer event has occurred or not
(since some of the etbr is cotransferred).
Your gel collapsed VERY early - well, why don't you put a little less
weight on it ? Last time I only had some 300g on it and the gel was
anything but pergamon-like even after 24h.
Of course, you should not try it before autumn.
Spring is a serious matter!
Good luck in your activities, whether hyb-bound or not,
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