We are currently experiencing difficulty using the 96 lane gel membrane
combs on our ABI 377s. We believe the problem is related to Ficoll as the
problem has only arisen recently with a new lot of Ficoll. Unfortunately,
new lots have not solved the problem. Has anybody else experienced this
problem recently? Even better, can any of you who have obtained consistent
sequencing results with 96 lane membrane combs summarize your procedure.
Suppliers of ficoll and any changes to the procedure described below would
be particularly helpful. I know we've been through this drill numerous
times but the problem has only popped up recently and is particularly
frustrating as we were obtaining consistent results prior to this.
Here are some specifics of our gels:
Sample prep: Edge Biosystems gel purification plates
Comb loading: MWG RoboAmp
Ficoll: 20% ficoll (Fisher and The Gel Company)
Gel: Long Ranger Singel packs
Procedure: Preheat gel to 51C. Add 1ml Ficoll to well, add comb and
buffer. Prerun for 30 sec. Thoroughly flush well and remove comb. Start
run.
Observations: Extensive shouldering and band compression
Thanks,
Michael Cariola
DNA Analyst II
Armed Forces DNA Identification Laboratory
1413 Research Blvd., Bldg. 101
Rockville, MD 20850
Office: (301) 319-0253 FAX: (301) 295-5932
e-mail: cariolam@afip.osd.mil <mailto:cariolam@afip.osd.mil>
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