DIG Southern: can Ethidium Bromide be a problem? - (Sep/28/2006 )
after figuring out a probe that gives me a reasonably nice band, I observe a very strong background signal that (after some thinking) might correspond to the Ethidium Bromide running front in my agarose gel. Has anybody experienced similar problems with DIG southerns? Should I simply reduce the EtBr concentration or is there some other trick to get around it (apply EtBr only to marker lane?)?
Thanks for your help!!
I've never had that problem, I don't think EtBr will casue much problem with a southern. You do destain (H2O or TBE/TAE for half an hour) after taking a gel image don't you? You don't need much EtBr to stain a gel, I usually use less than 1 microgram per mL of gel.
It would be very hard to apply EtBr to the marker lane only, short of chopping out the lane and staining it seperately.
Thanks for the comment. I actually don't wash the gel after taking the pic for the sake of washing, but there is one 10 min step of depurination (0.25M HCl) and 2x 15 min of denaturing (0.4N NaOH) followed by transfer. However, when I later check the membrane under UV I can (more or less clearly) see the Ethidium Bromide signal especially in areas of large amounts of DNA.
I was actually thinking of either chopping the marker lane and staining it separately (as you wrote).
when I do non-radioactive Southerns, I do not use EtBr (I use the set-up for gel-shift and I don't want interference with the DNA binding to the TF)
however, if I use blue juice at full concentration, I get a smudge across the blot at the location of the blue juice (this is not visible until the film is developed at the end). so, I modified the protocol...I dilute my blue juice about 1:10 and it's OK. the samples still go in the wells where they belong and the smudge is gone or lessened to the point that it doesn't obscure any bands
you may try this too? are you certain it's EtBr that you are seeing?