Facile recovery of individual high-molecular-weight, low-copy-number natural plasmids for genomic sequencing.

ABSTRACT: Sequencing of the large (>50 kb), low-copy-number (<5 per cell) plasmids that mediate horizontal gene transfer has been hindered by the difficulty and expense of isolating DNA from individual plasmids of this class. We report here that a kit method previously devised for purification of bacterial artificial chromosomes (BACs) can be adapted for effective preparation of individual plasmids up to 220 kb from wild gram-negative and gram-positive bacteria. Individual plasmid DNA recovered from less than 10 ml of Escherichia coli, Staphylococcus, and Corynebacterium cultures was of sufficient quantity and quality for construction of high-coverage libraries, as shown by sequencing five native plasmids ranging in size from 30 kb to 94 kb. We also report recommendations for vector screening to opt

SUBMITTER: Williams LE 

PROVIDER: S-EPMC1489313 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature