BAC sequencing

Vahe Bedian dnaseq at mail.med.upenn.edu
Tue Jul 9 04:34:18 EST 1996

Dear Fellow Sequencers,

We have recently been working on direct dye terminator sequencing of BACs
on ABI machines, and found it to be a challenge, inspite of the fact that
there are published reports of it. I thought I'd share some details of our
experience, hoping that some of you might find them helpful.

We found that using 2 ug of BAC template with 25 pmol primer and a 30 cycle
reaction, followed by our usual G50 spin column purification, and loading
all of the product onto a 377 gave excellent results, as long as the BAC
template was super clean. We were successful with a cesium chloride prep
that was very clean, using both vector and internal primers. We are now
testing to see if a miniprep that uses extractions will work.

On the 373 machines we were successful with 5 and 10 ug of the same
template, using the same reaction conditions. The big surprise here for me
was that even 10 ug of DNA, loaded into a single lane, did not give any
background yellow fluorescence problems that we have seen with 1-2 ug of
some phage and cosmid preps (presumably because they were not as clean).

If anyone out there in sequencingland has comments/alternate protocols,
please share.


Vahe Bedian
DNA Sequencing facility
Univ of Penn

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