painful gel loading???!!!!!!

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Thu Jan 30 15:14:32 EST 1997


At 08:43 1/30/97 -0500, Darren Natale wrote:

>Sorry about my rather severe understatement.  When I said "several" I
actually
>meant about 10 times, maybe more.  The buffer was TBE.  In my 12+ years
>of loading gels, this only happened one time, but it sure was interesting
>to watch!
>
>> Where I have had sample leave the wells was due to either ethanol,
>> isopropanol, mineral oil and the best for "tornadically" removing one's
>> samples from wells was phenol.  I would suspect one of these.
>
>Yes,  these would be the more common causes.  My "tornadic" (good
>description!) samples were PCR reactions (performed without mineral oil).
I loaded
>half the reaction onto the gel, only to have them float out in streams of
blue. 
>After a fresh buffer change, I loaded the remaining half without incident.

Darren:

Very interesting...  I also observed the problem many year ago when I was
trying a "zippy" prep.  It was a quick alkaline lysis (Solution 1,2,&3)
followed by an equal volume of phenol afterwhich a small portion was
directly loaded onto a gel without digestion(s).  It was a very quick way
of screening 20+ minipreps and aiding in what clones to focus on.  The idea
being that anything that did not display the supercoiled pattern of the
vector might indicate a clone to work up as it may have your insert of
choice.  Well... with phenol as the last step, it was common to end up with
some in the sample and that's where I saw the "Twister's" as some of my
sample simply "tornadoed" their way out of the gel.  I've seen some with
ethanol and isopropanol, but very little with mineral oil.  Once the
mineral oil goes to the top and "burps" upward, some but not all the sample
is lost...   

Back to the topic, I presume there is enough glycerol or ficoll to "weight"
the sample down?

David


=============================
 David L. Haviland, Ph.D.
 Asst. Prof. Immunology 
 University of Texas - Houston, H.S.C.
 Institute of Molecular Medicine  
 2121 W. Holcombe Blvd.  
 Houston, TX  77030 
 Internet:"dhavilan at imm2.imm.uth.tmc.edu" 
 Voice: 713.500.2413  FAX: 713.500.2424
 ------------------------------------------------------  
" Sometimes you're the windsheild, sometimes you're the bug."
=============================




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