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Plasmid Substrates
All our DNA substrates are available separately and have been prepared to a high standard for accurate quantification of results. They are guaranteed for use with all our topoisomerase assay kits, buffers and enzymes.
Click on the product headings below to view details and order online.
Supercoiled pBR322 DNA
Supercoiled plasmid pBR322 DNA is produced by the large-scale alkaline-lysis method ( Sambrook et al., 1989)1 from a high-copy derivative of pBR322 (Boros et al., 1984)2.
Negatively-supercoiled pBR322 DNA is further treated so that the DNA is more supercoiled and has a narrow range of linking numbers making it an ideal substrate for relaxation reactions.
Positively-supercoiled plasmid pBR322 DNA is produced by treating relaxed pBR322 with reverse gyrase before final purification.
It is shipped on dry ice at a concentration of 1mg/ml in TE (10mM Tris-HCl (pH 7.5), 1mM EDTA). Store at 4oC
Technical Documents
Relaxed pBR322 DNA
Relaxed plasmid pBR322 DNA is produced by the large-scale alkaline-lysis method ( Sambrook et al., 1989)1 from a high-copy derivative of pBR322 (Boros et al., 1984)2 and the resulting supercoiled plasmid is relaxed using topoisomerase I. This is further purified and optimised for topoisomerase assays
It is shipped on dry ice at a concentration of 1mg/ml in TE (10mM Tris-HCl (pH 7.5), 1mM EDTA). Store at 4oC
0.5 µg of relaxed pBR322 when incubated with 1 U of DNA gyrase in a reaction volume of 30 µl at 37oC for 30 minutes in incubation buffer is completely converted to the supercoiled form.
Technical Documents
Linear pBR322 DNA
Linear plasmid pBR322 DNA is produced by the large-scale alkaline-lysis method ( Sambrook et al., 1989)1 from a high-copy derivative of pBR322 (Boros et al., 1984)2.
It is linearised by incubation with EcoRI and further concentrated and purified to a final concentration of of 1 mg/ml in TE (10 mM Tris-HCl (pH 7.5), 1 mM EDTA). Store at 4oC
It is shipped on dry ice.
Technical Documents
Linking Number Plasmid
Plasmid pBR322 is partially negatively supercoiled to give specific linking numbers so that each marker has a range of 5-6 linking numbers. The kit contains a set of markers from relaxed plasmid covering approximately 23 linking numbers. Please enquire about single markers.
Technical Documents
References
- Sambrook, J., Fritsch, E.F. & Maniatis, T (1989). Molecular cloning: a laboratory manual,2nd ed., Cold Spring Harbor Press. Cold Spring Harbor, NY.
- Boros, I., Pósfai, G. & Venetianer, P. (1984). High-copy number derivatives of the plasmid cloning vector pBR322. Gene 30, 257-260
- Trask, D. K. & Muller, M. T. (1983) Biochemical characterization of topoisomerase I purified from avian erythrocytes. Nucleic Acids Res. 11, 2779-2800
- Shapiro, T.A., Klein, V.A. and Englund, P.T. (1999) Isolation of kinetoplast DNA, in DNA Topoisomerase Protocols Vol.I (Bjornsti, M-A and Osheroff, N., eds.). Humana Press Inc., N. Jersey, pp. 61-68
