?-easiest clean-up for sequencing

vilimf01 at mcrcr6.med.nyu.edu vilimf01 at mcrcr6.med.nyu.edu
Tue Feb 25 10:29:17 EST 1997


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.  I
tried a number of different kits without getting results that I liked. 
Then one day I picked up sequences from a new grad student and saw
that her sequences were far better than any of mine.  What was she
using? the old miniprep protocol from sambrook!  The protocol:

1.5ml overnight culture
resuspend in 100ul GTE
200ul soln II
150ul 3M KAc pH 4.8
Spin
phenol CCl3 (100ul will do) the supt - spin
CCl3 (equal vol) the supt - spin
PPt with 1ml EtOH - spin
wash pellet with 70% EtOH
Dry, and resuspend in 20-50ul TE with 20ug/ml RNase
send it off for sequencing

I didn't believe it at first, but it has been working great for me,
and now I will never do anything else.  Furthermore, you can't beat
the price.  But don't take my word for it, try for yourself.

					usual disclaimers	SVEN



In article <5eg25c$85e$1 at news.fas.harvard.edu>, tduda at oeb.harvard.edu (Tom Duda) writes:
> Does anyone have any easy methods for DNA clean-up (or not even cleaning
> up) for sequencing? We have been using everything from cleaning it up from
> gel cut-outs, to Qiagen, Geneclean, precipitations, etc. both before and
> after cycle sequencing...Are there any short-cuts?!
> 
> Thanks...
>  --
> T.F. Duda, Jr.               
> Biological Laboratories, Box #48 
> 16 Divinity Avenue
> Cambridge, MA 02138
> 617 496-8667  -- BL Rm 464



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