Bean Beetle Experiment
The experiment was divided into three days to test the detoxification activity of Beetles grown on Mung and Black-eyed peas. In day 1 of part I, four beetles per group were chosen from each food sources for both ANAE and BNAE activity. In part I, the crude extract was obtained by the experimental design outlined in the lab manual (Course Supplement for Bio 101, p.68). Each member of the group took care of one Bean beetle crude extract. First, the sex was identified by the following instructions of the lab manual on page 50. In addition, the methods for identifying sex were discussed in the introductory paragraph of this report. After determining the sex, we chose four beetles from the Mung beans food source and place them in separate 1.5 ml
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Next, transferred 200 uL of the crude extract to a set of microcentrifuge tube; the blank is created by adding 200 uL of the homogenizing buffer. Then 800 uL of pre-filtered dye reagent was added to each tube and vortexed for additional 10 seconds, followed by an incubation period of 10 minutes (Course Supplement for Bio 101, p. 71). We transferred 500 uL of the solution to the cuvette and measured the absorbance to 595nm. Using the BSA stock solution (2mg/mL=2000 ug/mL), we prepared the concentration of the protein standard by carrying out serial dilution at 2x, 5x, 10x, 20x and 40x. The 2x dilution was made by mixing 250 uL of BSA and 250 of the homogenizing buffer. The solution was then vortexed for 10 seconds. A similar procedure with different concentration of BSA and stock solution was carried out to find the other diluted concentrations. After the dilutions were made, 200 uL of the BSA concentration from the diluted tubes (2x, 5x etc.) were transferred to another microcentrifuge tube and 800 uL of filtered Bradford dye reagent was added to each microcentrifuge containing the BSA standard (Course Supplement for Bio 101, p. 72). The blank solution was created by adding 200 uL of homogenizing of the buffer. Then we incubated the tubes for 10 minutes and inverted 5 times before transferring 500 uL of the solution from each tube to the corresponding spectrometer cuvette; the absorbance was measured at 595 nm. The proper procedures were followed to discard the
Next, transferred 200 uL of the crude extract to a set of microcentrifuge tube; the blank is created by adding 200 uL of the homogenizing buffer. Then 800 uL of pre-filtered dye reagent was added to each tube and vortexed for additional 10 seconds, followed by an incubation period of 10 minutes (Course Supplement for Bio 101, p. 71). We transferred 500 uL of the solution to the cuvette and measured the absorbance to 595nm. Using the BSA stock solution (2mg/mL=2000 ug/mL), we prepared the concentration of the protein standard by carrying out serial dilution at 2x, 5x, 10x, 20x and 40x. The 2x dilution was made by mixing 250 uL of BSA and 250 of the homogenizing buffer. The solution was then vortexed for 10 seconds. A similar procedure with different concentration of BSA and stock solution was carried out to find the other diluted concentrations. After the dilutions were made, 200 uL of the BSA concentration from the diluted tubes (2x, 5x etc.) were transferred to another microcentrifuge tube and 800 uL of filtered Bradford dye reagent was added to each microcentrifuge containing the BSA standard (Course Supplement for Bio 101, p. 72). The blank solution was created by adding 200 uL of homogenizing of the buffer. Then we incubated the tubes for 10 minutes and inverted 5 times before transferring 500 uL of the solution from each tube to the corresponding spectrometer cuvette; the absorbance was measured at 595 nm. The proper procedures were followed to discard the