IUBio

dye blobs

Brigitte Wolf brigitte.wolf at akh-wien.ac.at
Tue Oct 3 12:46:50 EST 2000


To whom it may concern!

We have switched a few months ago to automatic sequencing with ABI
Prism 310. First, everything was perfect (or almost) but now, for no
explainable reason!!, there is hardly a probe that works.
We keep on having dye blobs, especially at the beginning of the
sequence (about 20bp long) so that the rest of the probe becomes
unreadable (signal is too low to be detected and analyzed properly).
These are the steps we have been using till now:
- big dye terminator cycle sequencing kit (2=B5l premix for 10=B5l total
reaction volume
- removing of the unincorporated terminator by precipitation with
isopropanol or by centrifuging (900g for 5 min) through sephadex G-50
- centrisep tubes work well but are inadequate when large quantities of
probes have to be analyzed

Who has a clue!?


Mag. Brigitte Wolf
AKH-Vienna, Department of Surgery
Research Laboratories
Waehringer Guertel 18-20
A-1090 Vienna
Austria

Tel.: ++43/1/40400/6848
Fax.: ++43/1/40400/6782


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