Fuzzy Sequencing Gel Bands

Brian Foley brianf at med.uvm.edu
Thu Feb 17 16:15:13 EST 1994


Steve Rodems (smrodems at students.wisc.edu) wrote:
: In article <01H8VS2QXCJ68WZ7PJ at MSSCC.MED.UTAH.EDU>,
: schoenfeld at MSSCC.MED.UTAH.EDU wrote:

: > Hi -
: > Can anyone tell me why I'm getting fuzzy bands on sequencing gels?  For
: > each sample, I usually do 2.5 hr and 6 hr runs side by side.  The 2.5 hr
: > run looks fine, but the 6 hr run is fuzzy.  Since they're on the same gel,
: > I don't think the fuzziness is due to something like gel temperature or
: > buffer depletion. Any ideas?
: > 
: Here's an idea, but this is only if your fuzziness is actually due to
: doublets.  If your sequencing primer contains any N-1 species (ie, 1 base
: shorter than its supposed to be) you can start to see doublets in the
: longer runs.  Minor amounts of contamination won't show up on short runs
: because you are looking at shorter products which don't contain as much
: label (if your labeling with an alpha-dNTP).  Longer runs will show your
: longer fragments which contain more label and thus your N-1 contaminants
: start to show.  This shouldn't be the case if you bought commercially
: available primers (eg, any Bluescript primers) but if you are having oligos
: made from a synthesis facility think about gel purifying them on PAGE. 
: Look closely at the fuzzy bands and see if they are actually doublets. 
: Hope this helps.  Good luck.

	I've not run into any primer problems like this.  I thought 
primers were synthesized in a 5' to 3' direction so that any N-1 primers 
would be lacking on the 3' end (no problem) not the 5' end (which would
cause this problem).

	My only guess is that perhaps you did not rinse the wells of the
comb before loading the later set?  The Urea leaching out of the gel will
then cause fuzzy bands.
	Other than that, I can say that I have never had good luck loading 
gels after too long of a pre-run.  If I need to read far from the primer
I always pour a second gel for the long run.  I have seen my gels
breaking down in a small region between where the upper buffer cools
the gel and the aluminum plate that distributes heat in the middle
of the gel.  My apparatus has a slight gap there where the gel overheats
and gets destroyed.

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



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