IBB Pune/17 August 2009

To check whether our IPTG was functioning properly, Sharvari performed a test for IPTG plus X-gal. According to the protocol provided by Sambrooke et. al. (for Screening Bacterial Colonies Using X-gal and IPTG:alpha-complementation), 40ul of 2% x-gal solution+IPTG was spread onto agar plates followed by untampered E. coli Dh5-alpha cells (100ul log phase). The following concentrations of IPTG were tested keeping X-gal quantities constant. This was done to check the least amount of IPTG required to observe expression in the cells in addition to chemical stability of our IPTG. Our IPTG was functional!!! The changing concentrations of IPTG did not show significant differences in blue color observed in the plates. Also this proves that the IPTG concentrations selected are more than enough to ensure protein expression. The patchy areas of blue rather than a matte may be due to just 40ul of X-gal being spread over the whole plate. This does not allow even dispersal of the chemical.