A method for plasmid copy number determination in recombinant Streptomyces
1992
Wrigley-Jones, C.A. | Richards, H. | Thomas, C.R. | Ward, J.M.
A method for the measurement of plasmid copy number in Streptomyces mycelia is described. It is based on the preparation (on a mini-scale) of the total DNA present in the mycelium followed by gel electrophoresis with ethidium bromide, photography and densitometric scanning of photographic negatives. The method is suitable for pIJ101-derived multi-copy plasmids and is shown to have comparable accuracy to similar methods developed for unicellular host organisms. A novel "copy number" reconstitution approach was employed to define the sensitivity of the method. We recommend the optimisation of plasmid topoisomer separation since we have found evidence for transient changes in their relative proportions for certain Streptomyces plasmids. Various plasmid topoisomers present in the extracts were identified by progressive DNaseI nicking of pure plasmid DNA.
显示更多 [+] 显示较少 [-]