spacing problems

Bruce Roe BROE at aardvark.ucs.ou.edu
Thu Aug 29 07:30:07 EST 1996


Ed,
        Somehow your gel concentration is not correct.  The ABI programs
require that the peak-to-peak spacing be in a very specific range that is
determined by the voltage and the acrylamide concentration (it's also effected
by urea concentration, gel thickness, and buffer concentration, among others).
This range is very very narrow and one little slip up and all is lost.
        There really is nothing you can do with this previous run except
manually try to make sense out of it.  The clue to your present problem is
that you are "using a new batch of acrylamide" and sorry to say but that's
the culprit.  Throw out all the reagents that are in question ( in this case
the acrylamide-bis) and re-make this solution taking upmost care.  Then, do a
run with some standard (pUC or m13) alone so you don't waste another batch of
valuable samples.  There really is nothing else to do except spend several
days doing standards with this new batch of acrylamide and varying the watts.
Sadly, a -12 could be that the run is either too fast or too slow and thus
you have to "guess" if you should increase or decrease the watts.  We, and
others, seem to obtain the "best" base calling when the range is between 9
and 10.
        Good luck,
Cheers......bruce
***************************************************************************
Bruce A. Roe, Ph.D    Department of Chemistry and Biochemistry
                      University of Oklahoma, Norman, OK 73019-0370, U.S.A.
Phone: (405) 325-4912 or 7610; FAX: (405) 325-7762; e-mail: broe at uoknor.edu
********************** http://dna1.chem.uoknor.edu/ ***********************

 You write: 
=> 
=> We ran a gel last night (on a 373) and although the gel file looks fine, 
=> the samples have a spacing of -12 !!!  yes, minus 12. As a result, the 
=> chromatograms look horrid and there are stacked bases that are
=> not been called, and essentially the 30 samples we ran are a total waste.
=> Again, on looking at the gel file, everything looked normal so we're a 
=> bit shocked. Has this happend to anyone before ?  we were using a new 
=> batch of polyacrylamide, but I doubt this would be the cause of the 
=> problem. Is it an analysis problem ? Is there a way to extract useful 
=> data from what appears to be a fine looking gel ? Anyone ever have such a 
=> weird spacing ? We've had problems before, but due to buffer problems, 
=> and these led to strange spacing calls, but the batch of buffer used on last 
=> night's gel has been performing well. We're puzzled and bewildered.
=> 
=> thanks for any and all ideas.
=> 
=> Ed Taboada
=> Dept of Biology
=> University of Ottawa
=> Ottawa, Canada
=> 
=> 
=> PS. please hurry !!! :)  we're going to run a gel with 4 samples that 
=> have sequenced well before as a test. But we'd hate to destroy a gelfile 
=> that may have had analyzeable data (the space left on the hard drive of 
=> the said computer would not allow us to keep both gel files).
=> 
=> thanks again.
=> 






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