In article <199511071943.NAA06477 at visar.wustl.edu>,
nikolaic at VISAR.WUSTL.EDU (Nikolai Chitaev) wrote:
> > To: methods-and-reagents at net.bio.net> > From: lc113 at mole.bio.cam.ac.uk (J.S. Good)
> > Subject: Qiagen plasmid kits
> > Date: 7 Nov 1995 17:03:38 GMT
> > Does anyone have any advice they can share on how to maximise the
> > yield from this kit? How can you get reproducibly constant yields?
> > It seems to be a matter of luck more than anything else.
> In our hands Qiagen spin columns (mini prep) works exelent taking into
> account that we work with low copy plasmids (ColE1 replicon) and with
> TG1 strain which is not the best.
> The same could be said about maxi-preps with Qiagen tip 500. 2.8-3.5
> mg/ml of plasmid is obtained routinly.
> P.S. Have no affiliation with Qiagen, just user.
> P.P.S Grow cells in TB overnight and use appropriate protocol suplied
> with the kit.
>> Good luck
Nikolai: But how many mls, do you get? From what I understand, the Q500
tip is supposed to be able to isolate 500 micrograms of DNA, with High
copy number plasmids that's usually less than 150 mls of a LB overnight.
I usually get 2 to 3 mgs per ml recovery, but thats in 200 microliters.
You can increase this if you reuse the columns (I usually split a 500 ml
overnight into 4 fractions and regenerate the column with QBT buffer
inbetween each fraction. From what Qiagen says you need to use the
column within 4 hours or so of the first equilibration. This has gotten so
labor intensive that I've gone back to CsCl preps on bugs grown in
superbroth when I need a lot of DNA. I may start trying some of the
massive prep kits that are available when I get a chance.
Stephen R. Lasky Ph.D.
Section of Experimental Therapeutics
Roger Williams Medical Center
Providence, RI 02908
Phone: 401-456-5672 Fax: 401-456-6569
e:mail: Stephen_Lasky at brown.edu