Growing the G Strain and Preparing the GCE (rGFP Crude Extract):
To grow the bacterial culture, use 10 ml of liquid LB growth media for incubation. 500 ml of the bacterial culture is allowed to grow overnight at 37°C. It is later shaken vigorously to increase the OD600 to 0.5, which means that time equals zero. At time zero, 1 mL of the culture is transferred into a 1.5 mL centrifuge tube and centrifuged for 5-10 minutes to obtain a pellet. The supernatant should be discarded. The centrifuge with the bacterial pellet is labeled “G0” and stored at -20°C. The culture is induced with 1 Mm of IPTG and allowed to keep growing. After 3 hours past induction, 1 mL of the culture is pelleted into a different 1.5 mL centrifuge tube, and the bacterial pellet is labeled “G3.” The centrifuge with G0 bacterial strain needs 15 mL of the strain to be collected and to be pelleted into the centrifuge tube. The last pellet is labeled “G3-15 mL.” Finally, both the G3 and G3-15 mL are to be stored at -20°C.
Preparing the rGFP crude extract:
500 ul of breaking buffer (made up of 10 mM Tris, pH 8.0 and 150 mM NaCl) is to be added twice to the G3-15 mL frozen bacterial pellet. After adding breaking …show more content…
Keep the solutions at room temperature for 10 minutes. Use the microplate reader is to measure the absorbance at 595 nm. The data collected needs to be plotted on a gridline-based graph and the standard curve is a line of best fit that is equally spaced in-between the highest and lowest data points. The Bradford assay is performed three times in succession (in triplicate) for Wash 1-6 and Elution 1-6. The absorbance value will be extrapolated from the standard curve created on the gridline-based graph. The total amount of protein in ug that was present in the 0, 2, 4, 6, 8, and 10 ug samples’ volume of BSA is to be