how to separate the nicked from of a plasmid from the closed form?
t_ro...@icrf.icnet.uk
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
Brian Foley
: does anyone know a method to separate the nicked circular form of a plasmid
In BioTechniques Volume 13 pages 550-554 (1992), Edward Hyman
describes a method for eliminating nicked plasmid by enzymatic digestion
with exonuclease I and exonuclease III.
This method results in a preparation of supercoiled plasmid with
no nicked plasmid. If you need nicked plasmid, with no supercoiled you
will have to find another method...
: Many thanks,
: T. Roldan
--
********************************************************************
* Brian Foley * If we knew what we were doing *
* Molecular Genetics Dept. * it wouldn't be called research *
* University of Vermont * *
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BrianC4208
writes:
>does anyone know a method to separate the nicked circular form of a
plasmid
>from the covalently closed circular form?.
Have you tried doing electrophoresis in 1% Agarose? This should
separate them, and then the bands can be cut out and the DNA can be
recovered by a commercial method for removing agarose like Geneclean.
Hope this helps.
Brian Cohen
Albany Medical College
Brian...@aol.com
Tracy Aquilla
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
aqu...@salus.med.uvm.edu
Zophonias Oddur Jonsson
You can separate nicked plasmids from closed circular on Ethidium Bromide
containing agarose gels. 50 ul EtdBr in E-buffer works fine. The nicked form
runs a lot slower than closed plasmids under these conditions since it can
bind a lot more of the dye. You cold then simply cut out the bands and elute
the DNA. I do however think that if you need large quantities of DNA a CsCl
spin would be more practical. If you don't overload the gradient and have
enough EtdBr present I can't see why it shouldn't work. Bauer and Viongrad
did it back in the sixties and a lot of people have done it since then!!
>Many thanks,
>T. Roldan
Good luck
Zophonias O. Jonsson
University of Iceland