how to separate cccDNA from nicked circular plasmid

churchillj at HAL.HAHNEMANN.EDU churchillj at HAL.HAHNEMANN.EDU
Tue Jun 7 17:06:58 EST 1994


Use extraction with acid phenol to purify cccDNA from a mix of nicked circular
and closed, circular coiled DNA.  Aubin R, Weinfeld M, and Paterson MC have an
extensive description in "Preparation of recombinant plasmid DNA for
DNA-mediated gene transfer"  on pp.3-13 of Methods in Molecular Biology, vol 7:
Gene Transfer and Expression Protocols, ed. by EJ Murray, 1991, Humana Press,
Clifton, NJ.

Essentially one dissolves the DNA in 50 mM NaOAc/75 mM NaCl, pH 4.0 to a
concentration of 250 ug/ml or less and extracts with an equal volume of phenol
that had been equilibrated with 50 mM NaOAc, pH 4.0.  The nicked circular and
linear DNAs partition into the phenol and only the cccDNA remains in the
aqueous phase (>95% cccDNA).  This technique yields plasmid that is transcribed
with high efficiency in Xenopus germinal vesicles.

Cheers!

Judy Rae Churchill, Dept. of Anatomy, Hahnemann University, Phila., PA
churchillj at hal.hahnemann.edu



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