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

Euan R. Taylor etaylor at ccu.umanitoba.ca
Mon Sep 20 17:12:42 EST 1993


In <1993Sep13.141945.1 at mcvax4.d48.lilly.com> dallas at mcvax4.d48.lilly.com writes:

>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".


I seem to remember seeing this in a plasmid blot and also a couple oftimes
in gels of radiolabelled primers. I assumed it was due to the radiation
level being sufficient to bleach the film. I dnt know exactly how that
would hapen. Inot convinced by the idea of overloading because the path
length of 32P is enough to blacken a good centimetre diameter of film in a
really strong signal, and the blank patches I have seen were very local
and discreet bands, course I also dont understand why path length wouldn't
make for a largely blurred and spread out pale area.
Any more ideas anyone?

-- 
Usual disclaimers. These are my own opinions and probably nobody else's.
etaylor at ccu.umanitoba.ca
"All great truths begin life as blasphemies"  George Bernard Shaw 



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