Pgex-Gfp Lab

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The first section of this experiment was to identify E.coli colonies containing the pGEX-GFPS65T for GFP fluorescence under blue light. Additionally, the transformed clones were amplified using PCR. The PCR products were run on 1 % agarose gel. To verify the transformation of the clones, restriction enzyme digestion was employed to cleave three different plasmids: pGEX-GFPS65T-1, - 2 and - 8, at specific sites. This was done with following restriction enzymes (RE): BamHI + PstI and EcoRI + PstI. A 1% agarose gel was run on the resultant fragments. The procedure for the experiment is outlined in details in the lab manual Laboratory Notes BIOC6017 Part1 (p.16-21). 2.2 Purification of recombinant GST-GFP A cell lysate containing the fusion protein GST-GFP, which was co-expressed with pGEX-GFP, was supplied. The purpose of this experiment was to purify GST-GFP and GFP from the bacterial lysate. The process was carried out according to the lab manual (p. 24) with following deviations.…show more content…
Hereafter, the supernatant was eluted and then washed twice with 1xPBS (phosphate-buffered saline). For resuspension of resin, 1 mL 1xPBS was added to the column. Next, using a Pasteur pipette 25 µL of the slurry was transferred to an Eppendorf tube for SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis). 5 µL thrombin was added to only half of the slurry solution. During this process, the pipette tip was accidentally lost in the column. When it was removed, small amounts of slurry and thrombin followed along. This can have influence on our results. Lastly, the column was incubated at room temperature on a rotating wheel. The group consisting of Abhilash and Aravind conducted the purification of a non-induced control. The procedure for this process is outlined in the lab manual (p.

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