Week 4

                This week we performed another NEON electroporation on a new plate of REF52 cells. The cell type was the same for this electroporation, as was the plasmid used (YFP-ER-E2F1); the only variation was the restriction enzyme digest that was performed. In the previous electroporation, we only linearized the DNA. This means that only a single cut was performed, turning a circular plasmid into a line of DNA. However, the linearized plasmid codes for unnecessary genes and shorter plasmids have higher transfection efficiency. We cut out an unnecessary drug resistance by performing a digest with two separate enzymes, and then performed an electroporation, hoping that this would result in more cells taking up the plasmid.

                In order to perform a NEON electroporation, cells have to be removed from the plate to which they are attached. A chemical called trypsin is used to cause the cells to detach from the plate. However, detaching the cells in this manner causes stress for the cells, and when trypsin is left on the cells for too long (a process called over-trypsinization) cell death may result. In order to avoid this, it is best to work quickly when trypsinizing cells, to remove as much trypsin as possible, and to make sure that the minimum amount of trypsin necessary is used.