Annotated Bibliography On Plasmid Purification - 1024 Words

Plasmid Purification Authors: Kashaf Baig Adam Jolly Abstract: Introduction: Plasmids are circular chromosomes that are found in bacteria (and other cells), can replicate independently (autonomous) and are separate from chromosomal DNA (Wikipedia, 2017). The plasmids have various conformation but the most important one is the negatively supercoiled conformation. (Wikipedia, 2017) also states that plasmids are suitable for an organism’s survival within the environment, as chromosomes generally contain all the necessary genetic information of an organism for normal conditions the plasmids on the other hand contain additional information which becomes useful in certain living†¦show more content†¦Then 30  µl of GelRed was added to the agarose-TBE solution and then mixed thoroughly. The gel was then poured onto a tray with a comb inserted to create the wells and was left to set for about 30 minutes. The 2nd method consisted of purification of the plasmid DNA provided (bacterial culture). 1.5 ml of 3 bacterial culture was added to 3 different centrifuge tubes and then centrifuged for 1 minute at about 8000 xg. The supernatant material from all 3 tubes was then discarded into 3% Virkon solution and the tubes were placed back in the rack. Then 250  µl of P1 Lysis Buffer was added to the tubes and vortexed followed by an addition of the same amount of Lysis Buffer P2 and mixed gently by inverting the tubes 6-7 times, the tubes were then left to incubate for 5 minutes at room temperature. 300  µl of Neutralisation Buffer P3 was added and then mixed thoroughly by inverting the tubes. All the tubes were now centrifuged at 11,000 xg for 5 minutes taking into consideration that the centrifuge machine was balanced when used to avoid any incomplete mixture. The tubes were then removed and 750  µl was carefully added to 1.5 ml spin columns as to not disturb the white residue (Na), the tubes were then again subject to centrifuge for 1 minute at 11,000 xg and the flow through discarded after the run. 500  µl of Wash Buffer PW1 was added and centrifuged for a minute (at the same xg), after