purification of 10 to 100 mg of plasmid?

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Fri Dec 11 04:13:58 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. 

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk



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