IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

Combs for Sequencing Gels <Question>

Brain Foley brianf at med.uvm.edu
Thu Aug 5 13:39:11 EST 1993


mwooten at ducvax.auburn.edu wrote:
: Since it works and it was the way we originally learned to do it, our lab
: has always used sharks tooth combs for loading sequencing gels.  Recently we
: tried using wells made with combs supplied with our BIO-RAD systems.  Loading
: was much easier (of course) but we got very poor results in all lanes.  This
: was true even when we loaded 2x as much sample.  

What type of poor results?  Fuzzy bands might be due to not rinsing the wells
out well enough just prior to loading.  Urea leaches out of the gel, into the
wells, and causes the sample to float on top of a thin layer of urea.

The other major factor is the distance between the wells.  With a sharks-
tooth comb, the samples nearly touch one another, whereas with a well-
type comb, there must be some distance between the wells.  THis has 
two disadvantages:  1) the bands on the autorad are a bit appart and thus
harder to read, harder to judge which one comes first.  2) the salt
concentraion of your samples may have local effects in the gel, causing
the lanes to run strangely.  When using a sharks-tooth comb, I often get
the best results if I load all lanes, the "empty" lanes between samples
are loaded with a reaction mix in which no radioactive lable was added, 
but is otherwise the same as a "real" sample.
: _______________________________________________________________________
: Michael C. Wooten                   
: Dept. of Zoology                    mwooten at ducvax.auburn.edu
: Auburn University   

--
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *



More information about the Methods mailing list

Send comments to us at biosci-help [At] net.bio.net