Mutangenesis (Continued)

On Wednsday (April 3rd, 2013), I continued working on mutangenesis in VvSTS enzyme with Namita. After we created several mutant plasmids by PCR last time, she added Dpn I to the solution to digest the original non-mutant DNA. Then, she did a biotransformation with a strain of bacteria (BW27784) that has the ability to ligate the new plasmids (because the polymerases actually form open-ended plasmids in PCR). Later, she sequenced a few transformed colonies in order to make sure that she had the right mutant before she did another transformation with another strain of bacteria for expression. In other words, the first transformation was to complete the circular plasmids and to check if the mutant plasmids have the right sequences. The second transformation was actually for cell expression.

Now came my task of the day: to create stock of mutant E.coli. There are 6 samples in total: control, T197I, T197A, T197M, et. all.

Namita provided six 15ml tubes. Since aeration is important in cell growth, a container can usually only holds 1/5 of its max. volume of solution. I put 3ml of cell media (3/15 = 1/5) and 3μl of antibiotics (1μg / ml) to each tube. Adding antibiotics is essential in making sure that the cells keep the mutant plasmids, which contain antibiotic resistant gene. Then, I pick one colony from each plate and add it to the media. Lastly, we put the tubes in incubator and let the cells grow a day, and then store them in -80C.

My job today was short and simple. Yet, precision was very important so I had to be careful in every step. I am looking forward to further discuss with Namita about the process:)

Where does this fit on our map?