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The following procedure is a modified version that we followed based upon the procedure provided by the Bio-rad Laboratories kit we used. (www.biorad.com) |
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| Preparation | Transformation | Purification | |
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The first task in our purification of the pGLO protein was to transform E. coli HB101 K-12 bacteria with a gene from the bioluminescent jellyfish, Aequorea Victoria, which codes for the green fluorescent protein (GFP). This involved several steps, including: Preparing agar plates Preparing ampicillin Preparing arabinose Ampicillin is shipped dry. 3ml of transformation solution was added to rehydrate the ampicillin via a sterile pipette. This was then mixed gently but thoroughly to ensure proper mixing. Arabinose is also shipped dry. 3 ml of transformation solution was added to the vial to rehydrate the arabinose, and the vial was mixed using a vortexer. The agar plate preparation took place in two steps. The first step was to make the solution. 500 ml of dH2O were added to a clean 1L flask and the contents of the LB nutrient agar were added. This solution was placed in the microwave for ~5 minutes, until the solution boiled and the agar dissolved. The agar was then allowed to cool on a stir plate to approximately 50˚C. The second step was to pour the plates. The lab bench was cleaned by washing with warm soapy water, rinsing well, and then rinsing with 70% ethanol. Once this was dry, 40 sterile plates were labeled, 16 as LB, 16 as LB/amp, and 8 as LB/amp/arab. LB agar was poured into the 16 plates labeled as LB to approximately 1/4 to 1/3 full. Hydrated ampicillin was then added to the media and the solution was mixed well. The 16 plates labeled LB/amp were poured to approximately 1/3 to ˝ full. Last, the rehydrated arabinose was added to the media and mixed. This was poured into the 8 plates labeled LB/amp/arab. These plates were then allowed to set at room temperature over the weekend to set up. They then were sealed with parafilm and covered with aluminum foil and stored in the refrigerator upside down to prevent condensation from dripping onto the agar. The next item on the agenda was to rehydrate the E. coli HB101. 250 μl of transformation solution was added to the vial containing the E. coli. The culture was allowed to sit for 5 minutes and then shaken to mix. The E. coli was then streaked onto a LB agar plate by inserting a sterile inoculation loop into the culture and streaking in the demonstrated manner. The plates were then stored overnight in the incubator upside down at 37˚C.
The pGLO plasmid was then prepared by adding 250 μl of transformation solution to the pGLO plasmid vial. This was then stored in the refrigerator until needed. See Colonies HERE. After colonies had grown on the plate, two colonies were individually scraped off of the plate. These colonies were transferred to individual microcentrifuge tubes with 250 μl of transformation solution. One loop of pGLO was added to one tube and the microcentrifuge tubes were put on ice for 10 minutes. The tubes were then heat-shocked at 42˚C in a water bath for 50 seconds to make pores to enable the uptake of the pGLO plasmid. Immediately after removal from the heat bath, the tubes were placed back on ice for ~7 minutes to close the pores. 250 μl of growth media was added to each tube and the cultures were allowed to sit at room temperature for 10 minutes. 100 μl of E. coli were pipetted onto one side of two plates containing LB/amp and one containing LB/amp/arab. These plates were then streaked by adding the E. coli containing the pGLO onto the LB/amp/arab plate and the LB/amp plate, and the E. coli containing no pGLO onto a LB and a LB/amp plate. The plates were then incubated at 32˚C for two nights. After incubation, the plates were tested for fluorescence, and thus the uptake of plasmid, by scanning them with a blacklight. Only the plates containing pGLO fluoresced under the blacklight. One colony was removed from each plate containing pGLO with a sterile loop and suspended in tubes according to whether the colony was grown with arabinose or without arabinose. These tubes contained 250 μl to LB broth to activate the gene and produce GFP in the culture that had not been grown in arabinose. To purify the GFP, the solution was transferred to microcentrifuge tubes were spun in the microcentrifuge at maximum speed for 5 minutes. The supernatant was poured into individual separate microcentrifuge tubes and the pellet and supernatant were both tested with the blacklight for fluorescence. The pellets fluoresced intensely, while the supernatant only glowed slightly. 250 μl of TE buffer was added to the pellet from the culture that had been grown originally in arabinose by choice of the experimenters. The pellet was resuspended by pipetting up and down with a pipette. A new pipette was used to add one drop of lysozome to the solution. This was mixed by flicking the tube. The lysozome’s purpose was to digest the bacterial cell wall. The solution was then placed in the freezer overnight. An HIC column was then prepared by setting up a stand so the column was level. The liquid was drained off the column and 2 ml of equilibration buffer was added to the column and drained to the 1 ml mark on the column. This was left until the next day. The next day, the frozen cells were spun at maximum speed in the microcentrifuge for 10 minutes. 250 μl of the supernatant was pulled off and transferred to a new tube. This solution glowed, meaning that it contained our protein. 250 μl of the Binding buffer was added to the supernatant and mixed gently. The HIC column was then drained until the buffer reached the surface of the matrix. A test tube was placed under the column to begin collection of solutions. 250 μl of the sample was added to the column and the output was collected in the first tube. The fluorescence remained at the top of the matrix, indicating it was not flowing through the matrix. 250 μl of wash buffer was added and collected in a second test tube. This solution glowed slightly, and the fluorescence had moved down the tube slightly. 750 μl of TE buffer was then added and the solution was collected in a third test tube. The fluorescence moved more quickly down the column and was present in the solution collected. These tubes were then covered with parafilm and stored in the refrigerator until needed for characterization. See the fluorescing tubes HERE. Back to Green Fluorescent Protein Homepage |
