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.
Thanks,
Vahe Bedian
DNA Sequencing facility
Univ of Penn
