purification of 10 to 100 mg of plasmid?

Brett Lindenbach brett at BORCIM.WUSTL.EDU
Fri Dec 11 12:03:08 EST 1998


>In article <74q0gi$ihu$1 at nnrp1.dejanews.com>,
>theodorn at medlib.georgetown.edu writes
>>Well, I had heard it about true silica, too, and just assumed it was also
>>true about the diatomaceous earth, because aren't diatom "skeletons" made of
>>silica? I thought that the differences between the two were due to the higher
>>surface area of the diatoms,
>
>I also thought that the case.
>
>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.


I agree that the problem will be getting good lysates.  Using CsCl and
a high copy plasmid, I'd expect to get at least 1mg/200ml culture (LB).
(My own record is ~5 mg). So, you could just spend a couple of days of
parallel purification on this scale and pooling them in the end.  I
second the recommendation of CsCl, since the quality of DNA can't be
beat.  I don't know what you're studying, but you'll probably want the
cleanest DNA for immunizing, assuming you want to invoke a response
against the gene rather than a contaminant.  Also, it'd seem to me that
if you have just way too many plasmids and/or amounts required, there
must exist a company that will handle purifications at these scales on
a contractual basis.  Who do the other DNA vaccinologists use?

Brett Lindenbach
brett at borcim.wustl.edu
Dept of Molecular Microbiology
Washington University School of Medicine





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