?-easiest clean-up for sequencing
Dr S.K. Coleman
sarahc at bc.ic.ac.uk
Fri Feb 28 11:50:39 EST 1997
On 26 Feb 1997, 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.
> Without fail, the CsCl material was THE best for transfection followed by
> the PEG Alk-lysis material. A distant third was material prepared by the
> Wizzard, and bringing up the rear was Qiagen. The Promega B-gal vector
> was prepared using all four methods.
> This is what worked for me, "in my hands" of course. But I think it does
> go to show that "kits" don't often do what they claim.
> David L. Haviland, Ph.D.
> Asst. Prof. Immunology
> University of Texas - Houston, H.S.C.
> Institute of Molecular Medicine
> 2121 W. Holcombe Blvd.
> Houston, TX 77030
> Internet:"dhavilan at imm2.imm.uth.tmc.edu"
> Voice: 713.500.2413 FAX: 713.500.2424
> " Sometimes you're the windsheild, sometimes you're the bug."
Hi - I'm afraid I have to disagree. For the last few years I was
regularly transiently transfecting HEK 293 cells. I originally
prepared the DNA by CsCl gradient x2. I then switched to the large
scale QIAGEN columns, the DNA obtained from the QIAGEN kits was as
good (in my hands) as that obtained by the old method - and a lot
faster. I found the QIAGEN kits to be very good and very reliable, and
for extraction of DNA from agarose or clean up after reaction better
than the Wizzard kits.
I havent used any of the other kits available.
s.coleman at ic.ac.uk
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