Large Scale Plasmid DNA Restriction Endonuclease Digest

[Song Tan]

In article <CKyMry.7rt at mentor.cc.purdue.edu> muriana at aclcb.purdue.edu (Peter M.
Muriana) writes:
>In article <2j8jfu$r0c at elna.ethz.ch>, tan at aeolus.vmsmail.ethz.ch (Song Tan) writes:
>  ..........stuff deleted........
>>As to your last question, we digest 50 - 200 mg of plasmid in one reaction
>>vessel several times a year (!).
>
>Just out of curiosity, what do you do with such large amounts of digested
>vector/plasmid DNA? - saving it for future use or selling it as a digested
>vector?  You mentioned **mg** and not **ug**, right?
>

Biophysical studies is the short answer.  

I know that digesting multiple mg of plasmid sounds like an inordinate amount,
but it's not when you're trying to isolate insert DNA to be used for
biophysical studies such as crystallography (see for example Richmond et al.,
Crystals of a nucleosome core particle containing defined sequence DNA, JMB,
199, 161-170, 1988).

I found a paper from 1981 which mentions preparing 350 to 1600 mg of plasmid
from 1000 g of packed cells (300 liter fermenter run), and digesting 250 mg of
plasmid with EcoRI !  (Hillen et al., Preparation of milligram amounts of 21
deoxyribonucleic acid restriction fragments, Biochemistry, 20, 3748-3756,
1981).  And I'll bet there are other examples in the literature.

An aside:  the Hillen paper mentions a nice way of fractionating DNA fragments
by PEG precipitation, even if the fragments have sticky ends.  PEG
precipitation will fractionate blunt-ended DNA fragments by size, but doesn't
work so well with sticky-ended fragments presumably because of interactions of
the sticky ends.  Addition of gelatin to 0.3% allowed fractionation of the
sticky-ended EcoRI fragments by PEG precipitation.

Song Tan
(Tim Richmond lab)
Institute for Molecular Biology and Biophysics
ETH-Honggerberg (Swiss Federal Institute of Technology)
8093 Zurich, Switzerland
email:  tan at aeolus.vmsmail.ethz.ch