So that I don't have to e-mail everyone who asked me for further information on
BigDye terminator cleanup. Please see Bruce Roe's web site at the following URL
for the microtiter G50 cleanup:
http://www.genome.ou.edu/protocol_book/protocol_partIV.html#IV.D.2
Regarding the EtOH/IpOH precipitation we did a comparison of this method with
the microtiter G50 cleanup and found them almost equivalent. However, the
ethanol precipitation gives about 75% of the signal strength of the G50 cleanup.
With the BigDye terminators you have enough signal strength that it usually
doesn't hurt your results though. The trick to getting the precipitation to
work well in a microtiter plate is to make sure you use 95%EtOH/5%IpOH and to
make sure your centrifuge spins fast enough to pellet the sequence ladders.
Our microtiter plate centrifuge is a Sorvall Super T21 and it can spin plates at
4300 rpm. It cost about $12000 though.
Hope this is useful. Please forgive me if I didn't respond to your e-mail
directly.
Bob
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Robert B. Chadwick
Director, Genotyping/Sequencing Unit
Division of Human Cancer Genetics
Ohio State University
420 West 12th Avenue
Columbus, OH 43210
e-mail chadwick-1 at medctr.osu.edu
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"Scientists have odious manners, except when you prop up their theory; then you
can borrow money from them." --- Mark Twain
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