DNA SEQUENCING

Dr. M.D. Jones mjones at uk.ac.crc
Tue Sep 7 04:24:46 EST 1993


Hi there fellow netters

I have a few queries about sequencing with the ABI 373A.  I have one and
am just getting to grips with large scale sequencing.

Question 1.  Has anybody got a reliable way of cleaning the plates to 
remove background fluorescence (apart from water and alconex)?  Any reliable
organic solvents?

Question 2.  Do people regularly perform a plate scan BEFORE they assemble
the plates and pour the gel?  Is it really necessary?

Question 3.  For sequencing short PCR fragments and small inserts, say up
to 300 base pairs maximum, what is the shortest time you can do a run?
Do you have to run it for 10 hours to get the data points for analysis?

Question 4.  I read with great interest the paper in NAR (vol. 21(15)
pp 3385-3390 [1993]) from Frederick Blattner's group on the M13 vector
JANUS, which allows inserts to be inverted by putting a M13 clone into
an E. coli strain expressing lambda int gene.  It sounds extremely useful.
Is the vector available?  Has anybody tried it out?  I have written to
Frederick Blattner, but am awaiting a reply.

Mick Jones
Department of Virology
RPMS
Du Cane Road,  London,  W12 0NN, UK
Tel: + 44 81 743 2030 ext. 2100
FAX: + 44 81 743 8331
Email: mjones at rpms.ac.uk



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