"Hole" in the middle of Southern bands?

Antje Haase antje at RT1.MENZIES.SU.EDU.AU
Fri Sep 10 17:44:02 EST 1993

>  Here's a weird problem: recently we have started seeing empty centers to
> intense bands in our PCR-Southerns (amplify, blot, probe with internal
> fragment, autorad).  Previous months of blotting never showed this.  Now
> past 2 weeks, several blots have it.  Sometimes only in certain lanes of a
> single blot, sometimes all.  Sometimes appears as a clear area in the lane
> (2 sides of band are hot), other times it's a "doughnut".  Anyone else
> ever seen this and/or opinions as to what's causing it.  BTW, We're using
> a StrataGene pressure blotter (have been for past year).  Thanks in
> advance...

I have seen this problem related to tiny little airbubbles between gel and
membrane that develop after assembling the blot. They are sometimes hard to
avoid. The only way is to prewet the membrane decently and also the first two
sheets of Whatman paper that go on the membrane.Then squeeze out any
entrapped air using a pipette.  I don't know the pressure
blotter but can imagine there could still be such a problem. The only other
reason I can think of may be finger (or glove) prints on the membrane.

Hope this helps


Antje Haase
antje at menzies.su.edu.au
Menzies School of Health Research

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