CsCl preps for ABI sequencing reactions

"Alexander Kraev" bckraev at aeolus.ethz.ch bckraev at wawona
Tue Mar 21 13:49:09 EST 1995


In article <jpcd0-2103951343150001 at macr1-3.welc.cam.ac.uk>, jpcd0 at mole.bio.cam.ac.uk (John Dixon) writes:
>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 have posted an article on dsDNA sequencing, which is now in the FAQ list,
but nobody seems to notice, the battle continues on... The problem of plasmid
sequencing is not reducable to a purification procedure, you may need to
change the strain, growth conditions and still get problems with 1 of 100
clones, which are "just difficult".
> 
>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 agree here, these columns worked well for me also, when I isolated DNA and
other people ran ABI/Taq sequencing with my preps...
>
>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)
>
Just what made you use the gradient? Was it low yield of the clone(s) or
something else?
For desalting you may have tried Pharmacia microspin columns, they are straight
forward to use, however, you may actually need to treat your DNA first with
a "Plasmid-safe" DNAse ( Epicentre; standard disclaimer applies ) and then
spin over such a column. This helped us with certain clones, which had
extremely low yield and hence were stark contaminated with chromosomal DNA.
 

*************************************************************************
Alexander Kraev, Ph.D.                 Internet: bckraev at aeolus.ethz.ch
Lab. of Biochemistry III               Phone: 0041-1-632-31-47
Swiss Federal Institute of Technology  FAX:   0041-1-632-12-13
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CH-8092 Zurich
"Some ideas are obscure not because they are complex, but because they 
 are excluded from our circle of comprehension" - Kozma Prutkov



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