purification of 10 to 100 mg of plasmid?

theodorn at medlib.georgetown.edu theodorn at medlib.georgetown.edu
Fri Dec 11 12:08:12 EST 1998


In article <1+SFOAAWJOc2EwFx at genesys.demon.co.uk>,
  "Dr. Duncan Clark" <Duncan at genesys.demon.co.uk> wrote:
> In article <74q0gi$ihu$1 at nnrp1.dejanews.com>,
> theodorn at medlib.georgetown.edu writes
<snip>
> Still doesn't solve the problem of purifying 100mg of plasmid. I too am
> going to have this problem in the next few months, maybe even in the
> gram range. I think CsCl is the way we will go.
>
> The biggest problem I see is getting the plasmid out of the bugs. It is
> not so easy to just scale up alkaline lysis protocols. The main problem
> is gently shearing the gloop from the lysed cells after addition of
> SDS/NaOH to be able to get it properly neutralised with Na.acetate.
> Depending on the plasmid 100mg could be 100litres of cells and therefore
> 1000g of cell mass! You can't lyse that sort of mass in less then a few
> litres and then it has to be eventually centrifuged. At some stage you
> have several litres of dilute plasmid. Too expensive to ppt with ethanol
> so maybe use propanol or better still PEG. Get the volume down to a few
> tens of mls then RNAse again, phenol, phenol/chloroform, chloroform
> extract then CsCl.
>
> I don't think Merlin is an option as I would think the losses could be
> quite high.
>
> Duncan

I think growing in Superbroth will get the volume of culture down to one or
two liters (for pUC or Bluescript, etc, type plasmids- 10 maybe for low
copy), but the cell mass will still be a problem, especially with SDS/Al
Kaline lysis. Maybe it's time to dust off the old lysozyme/Triton lysis
protocol here. Then treat the problem like you would when scaling up a
protein purification prep. Lyse, spin, Rnase, protease, then use an
anion-exchange column, which is a good step for concentration, too; you can
apply gallons to a column (or even batch absorb -  When I was an tech during
undergrad years we used to batch absorb from 16 liters of platelet extract,
then pour it into a 10 x 100 cm column for elution).  Then a gel-filtration
column to get rid of the RNA fragments, PEG and/or ethanol precip., and then
Bob's your uncle. But still test for endotoxin contamination.

I'm not sure CsCl will be practical with very large preps. And keep in mind
if this might be translated into human therapy, you really have to get the
ethidium out.

Nick Theodorakis

-----------== Posted via Deja News, The Discussion Network ==----------
http://www.dejanews.com/       Search, Read, Discuss, or Start Your Own    



More information about the Methods mailing list