Bromophenol Blue Experiment

To begin the experiment, 8 ml of 0.1% Bromophenol blue (BPB) solution was made by diluting 0.25% BPB solution. From the 0.1% BPB solution, six diluted solutions, ranging from 1:50 to 1:10000, were prepared. Each solution was then run in the UV spectrophotometer to measure the absorbance at 590nm. DI water was used as a blank and samples were measured starting from the least concentrated one. The graph of concentration versus absorbance was plotted from the obtained data. In the second part, each LB plate was inoculated with DH5α and DH5α pUC18, respectively. Two LB/Amp agar plates were treated in the similar way. The GVM agar plates was inoculated with A.fischeri. Between each streak, the inoculating loop was sterilized by placing above the
cone of the blue flame. After cooling, the loop was used to take an isolated colony.
Multiple streak lines were created across one quarter of the plate. In the second quarter, the sterile loop was streaked six times to create lines with some across the first set of streak. The procedure repeated with the third and fourth quarter of the plate. Agar plates with DH5α and DH5α pUC18 were placed in the 37o incubator and the plate with A.fischeri was put into the drawer with agar side up.The bacterial growth of each plate was latter recorded. In the third part , LB, LB/Amp, and GVM broth culture, were inoculated with DH5α, DH5α pUC18,and A.fischeri, respectively.The sterilize loop was put into the E.coli DH5α and then dipped into the LB broth. The loop was sterilized for next use, and the remaining broths were inoculated with similar technique. DH5α and DH5α pUC18 was incubated in 37o shaker and A.fischeri was put into the room temperature shaker. In the last part, the number of colonies grew in each LB plate of different diluted blank concentrations were determined. First, 0.1ml of DH5α culture was added into a sterile 9.9ml dilution blank. Next, 0.1ml of this solution was added to another sterile 9.9ml dilution
blank. Then, 1 ml of this solution was transferred into a sterile 9.0 ml dilution blank. The similar technique proceeded until getting 10-6, 10-7 and 10-8 of 9.0 ml dilution blank. From these last three dilutions, 0.1ml of each was added to each LB plate. The liquid was spread on the plate using sterilized spreading triangle. The triangle was sterilized by dipping in ethanol, putting into the flame and cooling for 15 seconds. After10 minutes, the plates were incubated at 370 C with agar side up.
Genomic DNA extraction from Aliivibrio fischeri :
First, five separate 1.5ml tubes were each filled with 1ml of A.fischeri MJ1, which was an overnight stockroom culture.
Another 5 microcentrifuge tubes were each filled with 1ml of overnight A.fischeri culture. The following procedure was used for one tube. The remaining nine tubes were treated in the similar technique. To begin, 1ml of overnight culture was centrifuged for two minutes at maximum speed (15000 x g) and the supernatant was discarded. Then, 600 μl of Nuclei Lysis solution was added gently to dissolve the pellet. Next, the tube was incubated for 5 minutes at 80oC and cooled to room temperature. Then, 3 μl of RNase solution was added and mixed with P-1000 pipette. The tube was incubated for 30 minutes at 37oC and cooled to room temperature. Next, 200 μl of Protein Precipitation solution was added to the tube and placed on ice for 5 minutes. After centrifuging the tube at max speed for 10 minutes, the supernatant was transferred to a clean tube with 600 μl isopropanol at room temperature. The liquid was mixed by vortexing the tube few times and centrifuged for 2 minutes. The supernatant was discarded. Next, 600 μl of 70% ethanol at room temperature was added and mixed gently. The tube was centrifuged for 2 minutes at max speed. Next, the ethanol was removed and the pellet was dried for 10-15 minutes. Then, at 40C, the DNA pellet was rehydrated in 25 μl of rehydration solution overnight. At the end, one microcentrifuge tube was the mixture of 5 tubes from the
stockroom culture, and another was the solution of the 5 tubes from the student’s culture.
Isolation of plasmids from E.coli :
First, to a 1.5ml microcentrifuge tube, 1ml of bacterial cells was added and then centrifuged at max speed. The supernatant was discarded. Next, another 1 ml of the cells was added, centrifuged following by removing the supernatant. The procedure was repeated until there were four centrifuge tubes, each having 5 ml of cells. Then, 250 μl of Buffer P1 was pipetted up and down to the tubes to disrupt the lump. The tube was latter inverted to mix after adding 250 μl of Buffer P2. Next, 350 μl of Buffer N3 was added and mixed by inverting for several times. The tube was centrifuged for 10 mins at max speed. The resulting supernatant was pipetted to QIAprep spin column. After centrifuging the tube for 60 seconds at max speed, 0.75 ml of Buffer PE was added to the column. The tube was centrifuged for 60 seconds. With flow through removed, the tube was centrifuged for an additional minute. Next, the column was put in a 1.5 ml microcentrifuged tube with no lid. Then, 40 μl Buffer EB was added to each column, waiting for 1 minute and centrifuged for 1 minute. The eluent from four centrifuge tubes were latter combined to a clean capped tube, which would be used to determine the concentration of DNA.