ABI 3100: red dye blob at 240 basecalls

Roland.Varecka at vie.boehringer-ingelheim.com Roland.Varecka at vie.boehringer-ingelheim.com
Fri Jun 29 06:24:57 EST 2001


Dear ABI3100 Users,=20

anyone got an idea how I could get rid of a huge, 10-15 peakbroad red=20
dye blob that shows up quite frequently in our sequence samples,=20
typically around 240 to 260 basecalls from start? This is particularly=20
annoying, since the appearance of this blob causes all other "T's" in=20
the remaining sequence to vanish. Only after manually shifting the=20
analysis start point past this blob is the software able to recalculate
the "T's" correctly.

We use the cycle sequencing conditions recommended by ABI.
Subsequently, the 10 uL reactions are diluted with ddH2O to 30 uL.
Diluted samples are immediately loaded onto overnight swollen and 1x
ddH2O washed Sephadex G-50 (Sigma) Multiscreen plates.
After centrifugation, samples are loaded directly into the ABI3100.

Interestingly, we see the red blobs grouped with certain labs (we
are a inhouse service lab, and frequently get the samples already after=20
each lab has run the Big Dye-CC reactions by itself).
However, on one day a lab may have perfectly clean sequences, and
the next day have red blobs in all samples.=20
So far I have not been able to pin down a pattern of the occurence.

Suggestins very welcome,


Sincerely, Roland
---------------------------------------------------------------
Roland M Varecka
Boehringer Ingelheim Austria GmbH
Dr. Boehringergasse 5-11
A-1121 Wien, =D6sterreich

Tel:=09xx43-1-80105-2392
Fax:=09xx43-1-80105-2360
Email:=09roland.varecka at vie.boehringer-ingelheim.com

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