Elimination of contaminant Escherichia coli chromosomal DNA from preparations of P1 artifical chromosome recombinants facilitates directed subcloning

The subcloning of large inserts (>50 kbp) from P1-derived artificial chromo somes (PACs) was found to be hindered by the presence of contaminating Esch erichia coli chromosomal fragments which, because of their smaller median s ize, are recovered preferentially as unwanted subclones. A significant frac tion of contaminating DNA was seen to persist after conventional plasmid pu rification methods. We describe a rigorous protocol for eliminating the bul k of contamination that involves plasmid isolation on commercially availabl e silica-based columns followed by three pulsed field gel electrophoresis s teps. Using this, we were able to subclone 55, 85 and 90 kbp PAC inserts bu t failed to subclone a 195 kbp PAC insert. After surveying a range of DNA p urification methods, we devised an optimised protocol that allowed us to su bclone the 195 kbp insert. The optimised protocol, which reliably yields DN A with essentially no contaminating material, consists of plasmid isolation on silica-based columns followed by treatment with highly purified DNasel and retrieval by electroelution of restriction-digested DNA electrophoresed on a single pulsed field gel. By inference it is applicable to the purific ation of large inserts from other single-copy plasmid vectors such as bacte rial artificial chromosomes (BACs).