?-easiest clean-up for sequencing
kraev at bc.biol.ethz.ch
Thu Feb 27 09:42:49 EST 1997
In article <220.127.116.1170226102642.00706cb8 at imm2.imm.uth.tmc.edu>,
dhavilan at IMM2.IMM.UTH.TMC.EDU ("David L. Haviland, Ph.D.") wrote:
> At 11:29 2/25/97 EDT, vilimf01 at mcrcr6.med.nyu.edu wrote:
> >I knew this would come up sooner or later. My answer is bound to piss
> >off many of the suppliers out there. We send our plasmids to a
> >core facility for sequencing, and I have been playing around with
> >different purification schemes to see which gives the best results.
> ** Edited **
> I'm not suprised by this in the least. One question though, this IS for
> automated sequencing, yes?
> Last year I was doing transient transfections in to COS cells. To optimize
> "things", I had to determine which method was best suited for transfection
> so I compared the standard CsCl (Maniatis), PEG Alk-Lysis (BioTechn.
> 14:532), Wizzard minis, and Qiagen midis. All rendered DNA that could be
> visualized and digested.
Both of you guys are right. Somehow the problem is always that way : is my DNA
pure or not pure? This is where the companies intervene and say: buy our
kits and get "ultrapure" DNA! Suitable for everything!! ( though not for taking
in, this requires FDA approval, which is lengthy and expensive:-). Probably
it depends on the educational background: in this newsgroup some persons
seem to be aiming at pure plasmid DNA in the chemical sense, others just
want methods that WORK for a particular application. I run an automated
sequencer and I do not use kits to have my plasmid DNA read to 1000 bases.
And it actually is VERY FAR from ultrapure ( a lot of RNA, at least). Now,
what really matters is which strain one uses to extract plasmid DNA.
If you start from JM101, C600 or similar vigorous old bugs, you will have a
hell of a time, going through all those precipitations, extractions, incubations
etc; take one of the slow bugs, e.g. XL-1, SURE (no affiliation to
any simple miniprep suffices for AUTOMATED sequencing. As for transfection,
we do try to make our DNA close to "ultrapure", because we think that eukaryotic
cells would not like to be stuffed by E.coli garbage, be it tRNA or proteins.
I hope this helps,
Alexander Kraev, PhD
Biochemie III, ETHZ Zurich
e-mail kraev at bc.biol.ethz.ch
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