how to separate the nicked from of a plasmid from the closed form?

Tracy Aquilla aquilla at salus.med.uvm.edu
Tue Jun 7 18:16:05 EST 1994


In Article <1994Jun7.171123.1 at icrf.icnet.uk>, t_rolden at icrf.icnet.uk wrote:
>does anyone know a method to separate the nicked circular form of a plasmid
>from the covalently closed circular form?. I have a mix which contain 50% of
>each form, when I do a CsCl gradient to isolate the closed form I always have
>contamination with the open form. does anyone konw a method to separare them,
>different from the CsCl gradient?.
>
>Many thanks,
>
>T. Roldan
>
    Well, the good news is that there is such a method; the bad news is that
the mechanism by which the procedure works is unknown. However, extraction
with acidic phenol will efficiently separate the supercoiled DNA (ccc) from
the nicked and linear forms. The pH of the phenol must be below 4.1; no
selective partitioning occurs at pH=4.2 or higher. This procedure is
included in the Erase-A-Base kit from Promega.
    Basically, you must first equilibrate the phenol with 50 mM Na+ acetate,
pH=4.0 (repeat until the aqueous phase is pH=4.1 or lower). Then you should
precipitate the plasmid DNA with 0.1 volume of Na+ acetate, pH=5.2 and 2.5
volumes of 100% ethanol. Recover the DNA and resuspend in ddH2O, then add 2
M Na+ acetate, pH=4.0 to bring the concentration to 50 mM, and 2 M NaCl to a
concentration of 75 mM (sodium concentrations higher than 125 mM will cause
the closed plasmid to partition into the organic phase!). Extract two or
three times with the acidic phenol, then once with chloroform:isoamyl
alcohol (24:1). Finally, recover the aqueous phase and add 0.1 volume of 2 M
NaCl and 2.5 volumes of ethanol to precipitate the supercoiled plasmid. It
is noteworthy that the DNA should not remain in contact with the acidic
phenol for too long, or excessive depurination may occur, and it is best to
do the extractions at 4C to avoid depurination. I hope this helps.
        Good Luck!
                    Tracy 
Tracy Aquilla, Post-doctoral Research Associate
Department of Molecular Physiology and Biophysics
University of Vermont, College of Medicine
aquilla at salus.med.uvm.edu



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