STEP 3: Renaturation:
5. Add 0.15ml ice cold Solution III. Mix by inversion and incubate on ice for 5mins. In this step, denatured DNA is renatured with solution III containing potassium acetate, glacial acetic acid. In this step small DNA (plasmid) renature back quickly whereas chromosomal DNA remained denatured.
6. Centrifuge at 12000g for 5mins and transfer the clear supernatant to fresh tube. Avoid contamination of white precipitate, even if you needs to leave 10-20µl supernatant.
7. Add RNase A to the supernatant at a final concentration of 20µg/ml. Incubate the tube at 370C for 30min. This step will degrades RNA present in the sample.
Figure 39.2: Steps in Plasmid Purification from bacterial culture.