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 supplied at a concentration of 1mg/ml in TE (10mM Tris-HCl (pH 7.5), 1mM EDTA). Store at 4^oC

0.5 µg of relaxed pBR322 when incubated with 1 U of DNA gyrase in a reaction volume of 30 µl at 37^oC for 30 minutes in incubation buffer is completely converted to the supercoiled form.

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References

1. Sambrook, J., Fritsch, E.F. & Maniatis, T (1989). Molecular cloning: a laboratory manual,2nd ed., Cold Spring Harbor Press. Cold Spring Harbor, NY.
2. Boros, I., Pósfai, G. & Venetianer, P. (1984). High-copy number derivatives of the plasmid cloning vector pBR322. Gene 30, 257-260