From: BACKD@QUCDN.QueensU.CA (Don Back)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: RE: Long Seq Gels
Date: 26 Jan 93 14:34:59 GMT
Distribution: bionet
Organization: Biochemistry, Queen's University

In article <1k2j0nINNt2q@menudo.uh.edu> bchs1b@Elroy.UH.EDU (Michael Benedik) writes:
>From: bchs1b@Elroy.UH.EDU (Michael Benedik)
>Subject: RE: Long Seq Gels
>Date: 26 Jan 1993 05:42:47 GMT

>In article <2B643AFA@adminbldg.lan1.umanitoba.ca>, GIETZ@bldghsc.lan1.umanitoba.ca writes:
>>------------------------------------------------------------------------------
>>I'm running fairly long (52 cm) sequencing gels with labelled DNA
>>fragments.  Right now we have to cut the top 10 cm because we don't have
>>X-ray film that's long enough.  I recall seeing X-ray film in rolls
>>somewhere.  Does anybody recall brand, size and price?
>>
>>Also, we don't have a gel dryer that accomodates long gels.  Any
>>alternative drying method?
>>
>>Cheers everybody
>>
>>Pablo A. Scolnik
>>scolnipa@esva.dnet.dupont.com
>>_____________________________________________________
>>
>>Pablo, 
>>    Why don't you try cutting the gel with a razor blade
>>and putting your gel to film in pieces.  If you load a sample
>>twice (at two different times) the place of the cut should
>>be no problem ( Overlap with the other load).  This worked 
>>for me when I was using King Kong gels.
>>
>>________________________________________
>>Dan Gietz
>>Department of Human Genetics
>>University of Manitoba
>>770 Bannatyne Ave, Rm 250
>>Winnipeg, Man, Canada
>>R3E 0W3
>>GIETZ@BLDGHSC.LAN1.UMANITOBA.CA
>>_________________________________________


>I agree with Dan that cutting the gel is probably the simplest. But
>long film does exist. A few years ago (I just counted and that means
>8 years) we were running 1 meter sequencing gels and had obtained 1 m
>long film and cassettes and even intensifying screens (the good old days).
>The film was standard kodak x-omat. So I would directly contact kodak or
>an appropriate kodak x-ray material distributor.


>----------------------------------------------------------------------
> Michael Benedik                                INTERNET: Benedik@uh.edu
> Dept. of Biochemical & Biophysical Sciences	
> University of Houston                          BITNET: Benedik@uhou
>-----------------------------------------------------------------------
When we run long sequencing gels (~90cm) we do one of two things depending 
on the label.
With 32P: leave the gel on the plate, cover with plastic food wrap, lay two 
sheets of film on the gel and overlap about 2 cm. Clamp the gel between two 
pieces of plywood, wrap in a black plastic garbage bag and place in the -70.

With 35S: The gel has to be dried. Carefully cut the gel in half with a 
sharp razor blade and a straight edge. Press a sheet of Whatman 3m onto the 
gel and peal off the gel, repeat with the other half. Dry and expose as 
normal. If you want to fix the gel and soak out the urea, place the gel 
over a sink/drainboard and flood with fix solution before transferring to 
the paper.
The most sequence you might loose is one or two bases, often you don't loose 
any. 

 
Donald Back                        
Department of Biochemistry         Phone: (613) 545-2982
Queen's University                 Fax:   (613) 545-2497
Kingston, Ontario. CANADA.         E-mail: BACKD@QUCDN.QueensU.CA
K7L 3N6