Fuzz bands on Genescan

glarson glarson
Thu Dec 19 01:01:04 EST 1996


Two problems are predominating our last 3 weeks of Genescan 377 runs:
	1. fuzzy (ie-poorly resolved) bands
	2. focusing of smaller bands (the width of smaller frags [~130b]on the
gel image is much narrower than larger ~350b] bands
Together these two problems combine to produce really inferior
images-needless to say how this affects the analysis.
What is the problem?
	Formamide-Ok checked it.  it was 5.5 then we replaced it with some
'good' stuff that pH'd @8.5 but the problem still persists.
Could the acrylamide (currently BioRad-not premixed) or urea be causing
these problems?
If you're wondering- yes we deionize the Acryl:BIS, yes we prerun the
gel as per directions.  No it not excessive salt in lane/multiplexed
samples since single loci under std PCR cndtns give the same result.
Our gel temps are running at 51oC, could a temperature reduction be
useful?
Would a change in acrylamide, BIS, urea, or TBE source be a good idea. 
WE just ordered Amresco reagents-identical to those which came with the
instrument in an attempt to rectify the problem.
Comments welcome
GLarson



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