Plasmid Prep Scale-up

[Roger Graham]

Netters,

When doing large scale plasmid preps using alkaline lysis (e.g. 50 g E.coli
wet weight) what is minimum volume of reagents required to sustain maximum
efficiency of plasmid recovery ? I'm sure scaling up directly from miniprep
amounts (1.5 ml culture pellet = 100 ul solnI, 200ul solnII, 150ul solnIII
of Birnboim and Doly procedure) would be overkill.

Thanx for your opinions.

--
Roger Graham
Dept. of Biochemistry
University of British Columbia
Vancouver, B.C., Canada
email : f...@unixg.ubc.ca

[Pendragon]

In article <flo-2207...@port57.annex4.net.ubc.ca>
f...@unixg.ubc.ca (Roger Graham) writes:

> When doing large scale plasmid preps using alkaline lysis (e.g. 50 g E.coli
> wet weight) what is minimum volume of reagents required to sustain maximum
> efficiency of plasmid recovery ? I'm sure scaling up directly from miniprep
> amounts (1.5 ml culture pellet = 100 ul solnI, 200ul solnII, 150ul solnIII
> of Birnboim and Doly procedure) would be overkill.

I don't know what your going to do with that much DNA as most work
requires a mere fraction of a minipreps worth. 50g of E. coli.?
Sounds like a fermenter run. Anyway we routinely get 5 mg of plasmid
DNA from 500 ml shaker cultures using the homemade Diatom maxi-prep
protocol. My guess is that the culture contains about 5-10 grams of
bacteria. Stick with the normal maxi-prep protocol amounts and split
your sample up into 4-6 fractions. Doing it all in one big prep could
prove unwieldy and it probably won't turn out as well.

Pendragon