By Jack G. Chirikjian
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Additional info for Biotechnology: Genetic engineering, mutagenesis, separation technology
Result After extraction of DNA, the success of the exercise is assayed by performing agarose gel electrophoresis on a portion of the extract. Plasmids typically exist as several forms: CCC (covalently closed circular) which is unnicked, supercoiled, compact, and runs ahead of other forms; OC (open circular) which has at least one single strand nick so that the ds DNA is allowed to relax from the supercoiled form; linear, in which nicks occur in both strands in the same vicinity so that the circular form opens.
Two procedures are presented: 1. A rapid isolation developed for small amounts (miniprep) of DNA which is not very pure but still suitable for some purposes; 2. A scaled up protocol for larger amounts of DNA of higher purity. This procedure calls for 2540 ml of an overnight culture. As proficiency develops, the culture volumes may be reduced to as low as 1 ml with proportionate reductions in reagents. Safety Guidelines Use gloves and goggles when handling the phenol:chloroform mixture. Phenol causes burns when in contact with tissue.
8. If it is necessary to remove contaminating RNA, the sample can be digested with RNase A. RNase A Digestion: Prepare a stock solution containing 10 mg/ml of RNase A. Heat in a boiling water bath for ten minutes to inactivate any DNase activity. Cool slowly. Store at -20°C. Add to a plasmid preparation to a concentration of 1020 µg/ml and incubate for 30 minutes at 37°C. Remove protein by 1:1 phenol:chloroform extraction followed by ethanol precipitation, as done previously. Centrifuge and dissolve the pellet in TE buffer or sterile ddH2O.