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

Remarks

• In the presence of IPTG, cells carrying a protein production plasmid do not grow because have dedicated all their resources to the production of the recombinant protein instead of cell maintainance.
• In the presence of the pLysS vector, IPTG also prevents colony formation except with certain vector (such as pET-3 and some vectors carrying the T7lac promoter). In the presence of pLysE, IPTG usually does not prevent colony formation unless the target protein is toxic.
  
  

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.

Protocol

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.

Reference: pET System Manual, 8th ed., 1999 (Novagen).

Last updated: 14 Nov 2000