CsCl preps for ABI sequencing reactions

John Dixon jpcd0 at mole.bio.cam.ac.uk
Tue Mar 21 08:43:15 EST 1995


In article <anderson-200395115201 at 128.252.181.14>,
anderson at pharmdec.wustl.edu (Eric C. Anderson) wrote:

> i was wondering if anyone had any information on using CsCl purified
> plasmids (both straight and desalted) for sequencing using the Taq
> terminator chemistry on an ABI autosequencer.
> 
 
I had big trouble about six months ago trying to sequence various plasmid
clones. I tried all sorts of different protocols for the plasmid preps,
but not in a very systematic way, ie I would try a different prep each
time the sequencing failed until I got one to work, and stick with this
until this failed. Eventually I got fed up with all this and admitted
that, as I had been told from the beginning, I would have to do CsCl
maxi-preps to be sure.

I never got one to sequence OK, and finally ABI tech help told me that the
preps ought to be completely desalted as the Taq is extremely sensitive to
salt conc. The idea of having to dialyse all my CsCl preps sounded just
too much hassle on top of ultracentrifugation etc so I've never tried it
since, and gone back to switching protocols until one works. FWIW in my
limited amount of cycle sequencing I found Qiagen plasmid preps the least
unreliable, but nowhere near guaranteed. 

I'd love to find one that worked every time, but it certainly wasnt CsCl
prep (maybe desalting would fix this, please let me know)

-- 
John Dixon                     Lab 44 (223) 334131
Wellcome/CRC Institute         Fax 44 (223) 334134
Dept Genetics
Cambridge University    
United Kingdom       e-m: jpcd0 at mole.bio.cam.ac.uk



More information about the Methods mailing list