Losel Baro, Nikita Damyan, Zhelah Guzeh
Over the last couple of weeks, we have finished working with the cloning of upstream region of the sprB gene and have begun work on the downstream region. The work we have been doing on the downstream region is very similar to that of the work we did for the upstream region, including running two confirmation gels, one to check if our downstream region was successfully inserted into our plasmid and a second gel later to see if any of our colonies were positive for the plasmid that we created earlier.
We started colony PCR after finishing up digestion, running a confirmation gel for our digestion to make sure our concentrations were good enough, ligation, and transformation. The first step in colony PCR was to streak every colony we could from the colonies formed from transformation. We ended up with five colonies that we streaked and incubated. In preparation for PCR, we used all five of our colonies and created a sixth one that we used as s control. Colony PCR was done to determine whether the plasmid was successfully inserted. The colony PCR was successful, and we were able to move on to work with colony PCR gel. For our colony PCR gel, we were able to confirm four positive colonies, but for further experiments, we only selected three, colonies 2, 4, and 5, with 2 and 5 having the brightest bands because they had a higher concentration. Colony 1 was the only colony we had that did not show a band in the colony PCR. We streaked colonies 2, 4, and 5 for isolation of pure cultures and plasmid preparation.
Overall, we have had a very smooth experience compared to when we worked with our upstream region, everything has been going successfully and we have had no problems in our way. The colony PCR gel was a huge success as we had four positive colonies. Our future plans are to start inoculation on our colonies for plasmid preparation and begin testing if both upstream and downstream regions are present in our final plasmid.