?-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 **
> 
> Hi:
> 
> 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
> 
> 
> =============================
>  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.K.Coleman

Biochemistry Dept
Imperial College
London

s.coleman at ic.ac.uk




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