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Re: technical problem



        Reply to:   RE>>technical problem

I just buy pre-poured ones :-)
Howard

--------------------------------------
Date: 1/3/99 10:26 pm
To: Howard Desmond
From: Michael Gribskov
I once experienced this problem when overlaying the running gel with
organic solvent,
i.e. isobutanol satuarated with buffer.  The solution: don't overlayer.


>On Mon, 1 Mar 1999, Grant wrote:
>
>> Can anyone help...?
>> 
>> 
>> 	Our institute is having problems running SDS page acrylamide gels. We have
>> found that we are losing proteins in the gels (as seen when using
>> prestained markers). When the gels are Coomasie stained it shows smeary
>> lanes with no defined bands. If the gels are transfered then there is a lot
>> of protein left in the gel. It seems to be the higher molecular weight
>> proteins which are most highly affected. 
>> 
>> 	We have changed the Water, SDS solution, Tris/Bicine stock solutions,
>> Acrylamide/Bisacrylamide solution and powder, APS and Temed. None of the
>> results show any consistency therefore we have not been able to narrow down
>> the problem(s). We have also used plastic wear instead of our own cleaned
>> glasswear as we thought that it maybe some residue retained on the
>> glasswear from cleaing that maybe affecting the gels. This has not been
>> reproducibly shown to make any difference. We also have experienced
>> variablility in wet and dry transfer but we think that this seems to be
>> down to problems with the gel and NOT with the transfer itself although
>> this maybe wrong. If you could provide any suggestions then we would be
>> very grateful as we don't know what else it could be. We use a continuous
>> gel system (Hoefer SE400 electrophoresis unit and transfer tank). The
>> recipe for the gel mixture and transfer buffer are given below:
>> 
>> 6% acrylamide gel:
>> 
>> 27.4mls H2O
>> 4mls 1M Tris/1M Bicine Solution
>> 8mls 30% Acylamide solution (37.5:1 Acrylamide:Bisacrylamide)
>> 400ul 10% SDS
>> 80ul TEMED
>> 200ul Fresh 10%APS
>> 
>> Run at 30-40mA for 5-6hrs (NOT cooled)
>> 
>> Wet Electrotransfer Buffer:
>> 
>> 7litres
>> 
>> 203g Glycine
>> 40.6g Tris
>> 20% Methanol.
>> 
>> Run at 200-250mA overnight (not cooled)
>> 
>> 
>> We have been using this system for along while and only now has this
>> problem arisen.
>> 
>> Yours sincerely,
>> 
>> G.Stewart
>> 
>> CRC Institue for Cancer Studies,
>> Birmingham University.
>> Birmingham. 



Michael Gribskov  -  http://www.sdsc.edu/~gribskov  -  gribskov@sdsc.edu
                               voice: 619.534.8312  -  fax: 619.822.0873


University of California, San Diego 
San Diego Supercomputer Center
Computational Biology Center, MC 0537 
9500 Gilman Drive
La Jolla  CA 92093-0537


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Date: Mon, 01 Mar 1999 13:08:45 -0800
To: G.S.Stewart@bham.ac.uk
From: Michael Gribskov <gribskov@SDSC.EDU>
Subject: Re: technical problem
Cc: kinases@sdsc.edu
In-Reply-To: <Pine.HPP.3.91.990301133645.157A-100000@fhs.csu.McMaster.CA >
References: <3.0.5.32.19990301142643.009053f0@cancer.bham.ac.uk>
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