Monday, December 5, 2016

CFU Assay #3

This time around we started our liquid culture with LB Broth. Slowly but surely we got to log phase (O.D. of  0.6nm - 0.8nm) which is the point where we want to induce our bacteria with IPTG so they will begin to produce our IL-8 protein. Also we didn't have any contaminated plates but we had two contaminated bottles of LB Broth that had to be discarded. We concluded as a class that the contamination came from the water bath where  we stored the bottles overnight. Even though we got to log phase something was still off about our data. Along with the O.D. numbers being off and not going up like we expected, a lot of the time point plates looked very weird and not normal. After talking to Dr. Matt Koci, and Mrs. Rizwana from NCSU Biotechnology Lab we realized we were using too much Kanamycin (KAN)  in the two assays before. This time we added 100ul of KAN per every 100ml of LB Broth. The concentration of KAN is 100mg/ml.

DATA HERE for Assay # 3 !!!

The samples we took from the liquid culture and put into the spectrophotometer was diluted by 5 fold. We had 800 ul of LB Broth into a cuvette and 200ul from the sample of the liquid culture. Meaning the time points 6- 24 that say <UNDILUTED did not have any LB Broth in them, it was all bacteria. To see all the time points in the undiluted form you just multiply them by 5.