In this experiment, we want to clone SOD gene into pUC19 plasmid of E.coli. Before ligating the target gene into the plasmid, the host cell needs to be competent to accept the vector. E.coli is not a naturally transformable bacterium thus; it needs to become transformable by chemical transformation. This technique is very useful to make competent cells that later will be used for transformation experiment in a short time. Actually there are not so much difference between chemically competent cells and competent cells prepared by electroporation. The only difference is the method to prepare it. Before adding the chemical, the cells need to be grown until the OD550 is between 0.3 and 0.5. At this reading, the cells are at its maximum viability. …show more content…
The plasmid needs to be isolated from the bacterial cells and the technique used is called alkaline lysis. This technique plays around with the pH to extract plasmid DNA. The culture is grown in medium containing ampicillin to select E.coli that have ampicillin resistance gene as their selectable marker. The addition of detergent to the cells causes cell lysis. The detergent attaches to the cell membrane and capture the protein and lipids of the cell membrane causing the cell to rupture. Then, the cell contents and DNA are released to the outside of the cell. The lysis buffer added causes the double stranded DNA in the cell to become single stranded DNA by disrupting the hydrogen bond between the bases. Next, acid is added to neutralize back the DNA to form double stranded DNA again. After centrifuge, the supernatant is collected as that is the plasmid while the pellet is the debris including protein and lipids. Protein and lipids are basically heavier than plasmid so, they settle down first before plasmid. The sample is centrifuged again to remove salt and debris. The pellet is the plasmid while the supernatant is the debris. Lastly, the plasmid is resuspended in TE and stored for DNA concentration