"Hole" in the middle of Southern bands?(More for less)

dallas at mcvax4.d48.lilly.com dallas at mcvax4.d48.lilly.com
Mon Sep 13 14:19:45 EST 1993


In article <GERLACHJ.54.747688503 at QUCDN.QueensU.CA>, GERLACHJ at QUCDN.QueensU.CA (Jim Gerlach) writes:
> In article <26nbu3$q7n at rigel.fccc.edu> hardy at mighty.fccc.edu (Randy Hardy) writes:
>>From: hardy at mighty.fccc.edu (Randy Hardy)
>>Subject: "Hole" in the middle of Southern bands?
>>Date: 9 Sep 93 13:43:31 GMT
>>
>> 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...
> 
> We sometimes see this as well and it is *NOT* caused by air bubbles. In our 
> hands it only occurs in bands that could be considered as "overloaded".


Any other theories?
>  
> Cheers,
> Jim
> -------
> Jim Gerlach                               Cancer Research Laboratories
> gerlachj at qucdn (BITNET)                   Queen's University
> gerlachj at qucdn.queensu.ca (InterNet)      Room A309, Botterell Hall
> tel (613) 545-6446                        Kingston, Ontario
> fax (613) 545-6830                        Canada K7L 3N6

When I first read this posting, I immediately considered overloading as well.
Sometimes if overloaded (and especially large fragments from a viscous
mixture), the DNA will stream down the walls of the well and less DNA in the
middle.  Also the 'donut' effect sounds like hybridization around the zone of
DNA where the concentration is less and may have actually bound to the matrix
better.  Bubbles could also be a problem but always in the band ?(OK I know
stranger things can happen).

Load less sample... as far as all the others weird things posted about
autoradiography... I haven't got the foggiest idea (pun intended).

Jim Miller
Lilly Research Labs
Indianapolis,IN



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