Week 3

                This week in the lab we performed our first electroporation on the REF52 cells. Electroporation is a process for getting either linearized DNA or plasmids into cells. Bacterial cells naturally will take up plasmids through a process called transformation, but mammalian cells, such as the REF52 cells we are working with, are unable to do this. Therefore, in order to get our desired plasmid into the cells, we need to open up the cell membrane in such a way that the plasmid can diffuse across. This is done by shocking the cells with a high voltage. This causes the membrane to become porous, and the large DNA molecules are able to easily enter the cell.

                We will be performing two electroporations because the effectiveness of the electroporation can change when the cell is linearized in different ways. Linearized plasmids, or plasmids that are cut so that the DNA is in a straight line instead of a loop, are able to enter electroporated cells more easily than their circular counterparts. The plasmid that we are working with contains some extraneous information encoded into it, and smaller plasmids are able to diffuse into the cells easier than larger ones. We have performed two separate restriction enzyme digest on the plasmid, and each will be electroporated into the cell to see which one is more effective. One cut only uses the enzyme ApaLI, and merely opens up the plasmid without removing any information. The other cut uses both ApaLI and MfeI-HF. This cut removes an unnecessary drug resistance from the plasmid in order to decrease the number of base pairs in the plasmid.