IIT Madras/28 July 2008

Continuing work on reviving the E. coli.K12z1 pLCFP strain that we received from Bangalore.

CFP measurements:

Sailaja and Gairik and Hemanth, along with some enthusiastic sophomores looking over, measured the fluorescence after inducing the pLac->CFP in the plasmid. The culture had been growing for 48 hours in 10ml of L.B. broth and Hemanth was sure it would have reached the death phase by now! We induced pLac->CFP by taking one ml out of three test tubes that had been growing and adding to it, 119.8 microlitres of 500mg/ml IPTG solution, to make the IPTG conc in the eppendorf roughly 0.25 mM. The fluorescence spec measurement was taken after 10-15 minutes of adding the IPTG. It was confirmed that the cultures taken from test tubes 2 and 3 had surely produced a lot of CFP!

KEY FACTS TO REMEMBER :

CFP excitation was done at 434nm. Emission measured at 470nm. The cuvette there needs a minimum of 2ml sample to give any measurement. Use water to dilute, if the volume available is lesser! Blanking was done with distilled water.

 Making Glycerol Stocks: 

Thus, decided that test tube 3, though, most probably well into the stationary phase, would be used to innoculate a 100ml LB broth in a conical flask. This was done and grown in an incubated shaker flask at 37 degrees. Gairik and Sailaja measured the OD after 2hours and 15 minutes of growth to be 0.101, looking to reach an OD of 1.0-1.5 (which is to be stored as a glycerol stock) in ten eppendorfs for later use. 60% glycerol made and autoclaved. 1% saline solution prepared for use as medium to resuspend cells and measure OD ,blanking done with 1% saline solution. OD measurement after 4 hrs of inoculation turned out to be 0.34 with the cells still in lag phase.The shaker flask with the culture was left in the shaker for 1 and half more hours with the OD being 0.45.Two and half hrs later, the OD reading of the culture diluted 2 times by saline was observed to be 0.78.(Effectively 1.56) Glycerol stocks were made by aliquoting 750 microlitres of glycerol and 750 microlitres of the culture (so that the final concentration of glycerol is around 30%) and stored at -40°C for further use. --Gairik 11:26, 28 July 2008 (UTC) --Sailaja 19:16, 29 July 2008 (UTC)