An easy and accurate agarose gel assay for quantitation of bacterial plasmid copy numbers.
An assay for quantitation of plasmid copy numbers in bacterial cell cultures has been developed and validated. The method combines isolation of total bacterial DNA (including both plasmid and genomic DNA), running a series of twofold dilutions of total DNA in an agarose gel followed by ethidium bromide staining, and subsequent scanning of the gel picture negatives. We have developed a novel set of rules for integration of the scan data that allows us to achieve high assay precision, accuracy, and sensitivity. The assay validation results were as follows: intra- and interassay precision with %CV of 8.2-9.9 and 7.1-9.8%, respectively; ruggedness with %CV of 9.3-17.5%; spike recovery of 80-102%; and sensitivity of 1 plasmid copy per genome.
Chiron Corporation, 4560 Horton Street, Emeryville, California 94608-2916, USA.
E A
EA
Pushnova
M
M
Geier
Y S
YS
Zhu
D004269:DNA, Bacterial / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D004996:Ethidium / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D018628:Gene Dosage; D005821:Genetic Techniques; D007376:Interleukin-2 / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D007376:Interleukin-2; D004996:Ethidium