Week 6

                This week in the lab, we ran into a major problem. Before drug selection, we checked the cells under a fluorescent microscope, and yellow fluorescence was present. We then checked the cells under a regular microscope during drug selection, and saw patches of drug resistant cells forming. However, when we checked the patches this week under the fluorescent microscope, the patches did not fluoresce like we expected. There were only a few cells fluorescing across 12 p100 plates, and one 6 well plate. Such a small number of fluorescent cells bodes ill for trying to form a single cell colony, as we were hoping for a substantial number of glowing cells to sort using flow cytometry.

                The solution that we determined for this problem is twofold. First, we are going to sort the cells that we have already transfected using flow cytometry anyways. The flow cytometry machine tests for glowing cells and has the capability to sort them, either into a microcentrifuge tube or a 96 well plate. We are hoping that the machine will detect more fluorescence then we were able to see with the microscope. We will sort the glowing cells into a microcentrifuge tube and then replate them and let them grow out before re-plating in a 96 well plate.

                We also decided to perform a second and third transfection, each at two voltages. We performed the transfection with both uncut DNA and DNA cut with MfeI-HF. The voltages used were 1900V and 2100V. No sparks were observed. We hope to get better results than in the previous transfection, and the newly transfected cells will provide a backup plan if the flow cytometry fails.