Yesterday we created recombinant DNA by cutting two plasmids with two enzymes, which was used in today's lab. After a time of incubation in both cold and hot water, we tested our DNA in an electrophoresis gel to determine whether our DNA was completely digested. The results of my gel said that one of the plasmids was not completely digested, but with the DNA incubated during the time of the gel analysis, I assume that it was enough time to have complete digestion. Then I added some DNA ligase to bond the cut plasmid together, which was the recombinant DNA that we were working with today.
We also prepared a culture of E. coli cells. It was not a long process to prepare, but it took a long time to get the cells to the ideal culture density. After getting the ideal density, we had several treatments of CaCl and then incubation to prepare the cells for transformation—this preparation increases the efficiency of the transformation ten-fold because it neutralizes the negative membrane. With the incubation the DNA is facilitated into the cell.
In the continuation of our lab from the previous day, we transformed some cells with our generated recombinant DNA. It was a long process with a lot of waiting. I am concerned if the cells will have grown by tomorrow because I would have to start the lab over, which is the equivalent of one day plus whatever we would be doing the day I reconstruct my culture.
Later in the week, we will get to see the results of our transformation and test whether or not our transformation was successful. We will test the cultures by pipetting antibiotics into the cultures. The plasmid encoded for antibiotic resistance, so to be successful the transformed cells should survive in an environment with antibiotics. Before we test our cultures, we will be analyzing our own blood samples.