Very large Plasmid Preps

[tan at aeolus.vmsmail.ethz.ch]

In article <flo-170694164305 at cellsi.nce.ubc.ca> flo at smtp.unixg.ubc.ca (Roger
Graham) writes:
>What protocol is best for doing very large plasmid preps in E. coli, e.g.
>>200 g wet cell weight ? Do people just keep scaling up alkaline lysis ? Is
>there a more efficient way ? Thanks for input.

Don't know if this is the best method around, but it works.  Yes, it's
essentially a scaled up alkaline lysis, with a Sephacryl S1000 column stuck in
at the end to remove any RNA and chromosomal DNA leftover.  Details for the
prep starting with 150 g of wet weight cells can be found in:
Richmond, T.J., Searles, M.A. and Simpson, R.T., Crystals of nucleosome core
particle containing defined sequence DNA, J Mol Biol, 199, 161-170 (1988).


Song Tan
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