|Plate||cells that grow on these plate|
|LB plate||all viable cells|
|LB plate + antibiotic||cells that still carry the plasmid|
|LB plate + IPTG (1 mM)||cells that have lost the plasmid or mutants that have lost the ability to express the target gene|
|LB plate + antibiotic + IPTG (1 mM)||only mutants that retain the plasmid but have lost the ability to express the target gene|
In a typical culture useful for producing target protein, almost all cells will form colonies both on the LB plate and on the LB plate + antibiotic; less than 2% of the cells will form a colony on the LB plate + IPTG; and less than 0.01% will form a colony on the LB plate + antibiotic + IPTG.
With unstable target plasmids, the fraction of cells that have lost the plasmid will be reflected by an increase in colonies on the LB plate + IPTG and a decrease on ther LB plate + antibiotic.
|1.||Immediately before induction with IPTG (at OD600 is approx. 0.6), take a 100-ml aliquot of the cell culture.|
|2.||Make a serial dilution of the cell suspension, including a 105 and 106 dilution.|
|3.||Plate cells at a dilution of 105 on the LB plate + IPTG and on the LB plate + IPTG + antibiotic.|
|Plate cells at a dilution of 106 on the LB plate and on the LB plate + antibiotic.|
|4.||Incubate the plates overnight a 37°C.|
|5.||Count the number of colonies on each plate.|
Last updated: 14 Nov 2000