Elimination of contaminant Escherichia coli chromosomal DNA from preparations of P1 artificial chromosome recombinants facilitates directed subcloning.
The subcloning of large inserts (>50 kbp) from P1-derived artificial chromosomes (PACs) was found to be hindered by the presence of contaminating Escherichia coli chromosomal fragments which, because of their smaller median size, are recovered preferentially as unwanted subclones. A significant fraction of contaminating DNA was seen to persist after conventional plasmid purification methods. We describe a rigorous protocol for eliminating the bulk of contamination that involves plasmid isolation on commercially available silica-based columns followed by three pulsed field gel electrophoresis steps. Using this, we were able to subclone 55, 85 and 90 kbp PAC inserts but failed to subclone a 195 kbp PAC insert. After surveying a range of DNA purification methods, we devised an optimised protocol that allowed us to subclone the 195 kbp insert. The optimised protocol, which reliably yields DNA with essentially no contaminating material, consists of plasmid isolation on silica-based columns followed by treatment with highly purified DNaseI and retrieval by electroelution of restriction-digested DNA electrophoresed on a single pulsed field gel. By inference it is applicable to the purification of large inserts from other single-copy plasmid vectors such as bacterial artificial chromosomes (BACs).
Davidson
H
H
MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK. heather@hgu.mrc.ac.uk
Doherty
A
A
Stevenson
B
B
Boyd
A C
AC
Porteous
D J
DJ
D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D004274:DNA, Recombinant / Q000032:analysis* / Q000302:isolation & purification; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D010957:Plasmids / Q000235:genetics
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D004262:DNA Restriction Enzymes