CRISPR is the process of gene modification, in this case, a deletion. We received the Px458 plasmid from a manufacturing lab, where the guide RNA targeting IL-22 was cloned (Figure 3). This plasmid contained a coding region for GFP which acts as a reporter when a plasmid is taken up by a cell. The GFP is a gene that codes for green fluorescence when exposed to 395nm light. The purpose of the GFP is to identify successfully modified 4T1 cells for separation. We also have a control called a “gene desert” which is the same Px458, but codes for a guide RNA that is devoid of genes in the mouse genome. 
Plasmid DNA needs to be purified in order to achieve the ideal concentration, using plasmid midi-prep. This was achieved by adding Buffers P1 and N3 and then centrifuging for ten minutes at 13,000. Then, the supernatant was applied to a QIAprep spin column and centrifuged for one minute. The spin-column was then washed with Buffer PE and centrifuged for another minute. The flow-through was discarded and the spin column centrifuged for another minute. To elute the DNA the QIAprep column was placed in a microcentrifuge tube and water added to the spin column. It was allowed to stand for a minute and then centrifuged for another minute. Afterward, the concentration of the purified DNA was measured, with good purifications with a concentration of around 150nM.
Transfection is the insertion of new nucleic acids, aka DNA, into eukaryotic cells. In this case, the PX458 plasmid was inserted using lipid-mediated transfection. The transfection procedure is when a cationic lipid transfection reagent mixes with the desired plasmid to produce liposomes that enter a mammalian cell to deposit the plasmid in the cell. If transfection is done correctly, the cell line will express modified genes from the plasmid. LypoD was tested as a reagent to enable the plasmid’s entry into the 4T1s, with no results. Other reagents were also tested in order to find the best and most effective reagent; in the end, lipofectamine 3000 proved to have a high efficiency and expression results for plasmid transfection. There was also a control in which no modified DNA was added, but the cells still went through the same process, including the reagents and midi-prep. Using this control helped confirm that the reagents were not affecting the process and that the cell sorter and flow cytometer were only identifying the modified cells.