2.4. DNA Preparation

QIAGEN Plasmid Mini Kit was used to prepare up to 20 μg of high-copy plasmid DNA. A selective plate was streaked followed by the selection of a single colony. LB medium was used as a starter culture to be inoculated containing the appropriate selective antibiotic. This was followed by the incubation with vigorous shaking for approximately 8 h at 37°C; 3 ml selective LB medium was then used to dilute the starter culture. The culture was left in the incubator at 37°C for 12–16 h. To harvest the bacterial cells, centrifugation was done followed by the resuspension of the formed pellet with shaking. Subsequently, 0.3 ml of Buffer P2 was added and mixed thoroughly by vigorously inverting the sealed tube 4–6 times. Later, the tubes were centrifuged, and the supernatant was promptly removed as it contains the plasmid DNA.

1 ml Buffer QBT was used to equilibrate a QIAGEN-tip 20, the column could empty by gravity flow, and then the supernatant was applied to the QIAGEN-tip 20 and, by gravity flow, it entered the resin. Buffer QC was used to wash the QIAGEN-tip. This was followed with the elution of the DNA with 0.8 ml buffer QF, and then isopropanol was added to precipitate it. It was mixed and centrifuged immediately then the supernatant was carefully decanted. Ethanol was used to wash the DNA pellet and then centrifuged again, and the supernatant was carefully removed as to not disturb the pellet. Finally, the pellet was air-dried, and the DNA was redissolved in a suitable volume of buffer. Bot spectrophotometry at 260 nm and quantitative analysis on an agarose gel were used to calculate DNA concentration as to determine the yield. A260 readings should lie between the values of 0.1 and 1.0 to judge the reliability of spectrophotometric DNA quantification.