High plating density improves Big Blue system efficiency without loss of sensitivity.
To increase efficiency in the Big Blue system, the plating density was increased from 15000 to 30000 or 45000 plaque forming units (pfus) per plate by increasing the density of the E. coli lawn and decreasing individual plaque size. Small plaque size ensured minimal overlap of the plaques. Liver from one 3- and one 25-month-old mouse (low and high mutation frequencies, respectively) was analyzed and neither plating density nor plaque size affected mutant/mutation frequency and pattern. The color intensity of particular mutant plaques was not affected by plaque size or plating density. Optimal sensitivity is achieved by sequencing mutants to calculate the mutation frequency from the mutant frequency and to identify altered patterns of mutation. Detailed effort and cost accounting of the Big Blue system (including mouse handling, DNA extraction, plaque screening, plaque purification, and DNA sequencing) reveals that one-quarter of the total effort is devoted to plating and screening of plates. This effort is reduced two fold with high plating density. The total cost of the Big Blue system is reduced by 17%. The total cost of the High Plating Density Big Blue system is now only 12% more costly than a selectable assay and offers an extensively validated system with a large mutation database representing a decade of effort.
Heinmöller
P W
PW
Department of Molecular Genetics, Beckman Research Institute, City of Hope, Duarte, CA 91010, USA.
Hill
K A
KA
Sommer
S S
SS
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D029968:Escherichia coli Proteins; D005260:Female; D056968:Lac Repressors; D051379:Mice; D008822:Mice, Transgenic; D009152:Mutagenicity Tests; D012097:Repressor Proteins / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D012097:Repressor Proteins