SHOTGUN SEQUENCING

Alex Knight alex at mrc-lmb.cam.ac.uk
Tue Dec 7 05:57:59 EST 1993


In article <1993Dec7.084144.4413 at crc.ac.uk> Dr. M.D. Jones,
mjones at crc.ac.uk writes:
>Does anybody out there have a detailed fool-proof 'sonication' protocol ?
>Anybody have any advice/tips/etc on how to get the best out of this
procedure ?

I wouldn't like to give a detailed protocol, as it really depends on what
machine you have. But the key thing is not to overdo it. The original
Bankier paper (Bankier et al. (1987) Methods Enzymol. 155, 51-93) advised
2X80s, and the collection of two size fractions, 300-600 and 600-1000bp.
However, they advised me when I was having problems to use 3X10s, and to
collect one size fraction e.g. 500-1000 bp. There is no need to get
really small inserts, especially if you get reads of 300+. You should be
able to assess the results of your sonication by running gels, and then
select the minimum possible time. I think that keeping the DNA cold is
also important.

I know from my frustrations in the past that sonication is the single
most critical step in the shotgun procedure. But when it works, you get
great data!

Good Luck,

Alex
alex at mrc-lmb.cam.ac.uk



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