Fuzzy Sequencing Gel Bands

Steve Rodems smrodems at students.wisc.edu
Mon Feb 14 22:07:00 EST 1994


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.

-- 
Steve "Some day I will get the hell out of Wisconsin" Rodems

"You know, the other day I ... oh, wait, that wasn't me"



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