![if !IE]> <![endif]>
The method of plasmid isolation by alkaline lysis method was originally developed by Brinboim and Doly (1979). To isolate the good quality and quantity of plasmid the population of bacteria is important and growth phase. The bacterial cells are grown for log phase (0.4-0.6 O.D) at 600 nm in an appropriate medium and in appropriate conditions. Luria broth is congenial for E. coli DH5α cells, and 2xTY for E. coli TG1 cell. The bacterial culture is grown at 37oC in shaking incubator at 200 rpm for overnight. These bacterial cells are lysed with alkaline Solution-I (Glucose 50 mM, Tris-Hcl 25 mM and 10 mM EDTA pH 8.0) and freely released protein and genomic DNA is denatured by adding alkaline Solution-II [0.2 N NaOH, 1% sodium dodecyl sulfate (SDS)]. NaOH, increases the pH and facilitates for denaturation, in contrast to the pH is decreased by adding the alkaline Solution-III (5 M potassium acetate- 60 mL, Glacial Acetic acid- 11.5 mL and sterile water-28.5 mL). This solution renature the plasmid alone but the genomic DNA will be linear and because of its larger size it unable to renature in short time of incubation. Hence, the denatured DNA precipitates and removed from the solution. The plasmid from the solution is precipitated by isopropanol and dissolved in sterile water/ T10E1 buffer. The detailed protocol discussed below.
Inoculate the individual colonies in 1 mL of LB with appropriate antibiotic selection pressure and incubate the cultures at 37oC in 200rpm. The overnight grown cultures are centrifuged at 10,000 rpm for 1 min at 4oC. The supernatant are removed and the pellet is washed with 250 µl of STET. The suspended cells in STET buffer are centrifuged at 10,000 rpm for 1 min at 4oC. Collect the pellet and dissolve in 100 μl of ice-cold alkaline lysis solution-I by vigorous vortexing. Later, add the 200 μl of freshly prepared alkaline lysis solution-II to each tube and the contents are mixed by inverting the tubes for 4 to 5 times and keep it in ice for 5 min. To this suspension, add the ice cold 150 μl of alkaline lysis solution-III and again mixed thoroughly by gently inverting the tubes for 4-5 times. Keep the tubes in standing position for 3 min. and spin for 10,000 rpm for 10 min at 4oC. The supernatant is needed to transfer to fresh tube and add 2 μl RNase (10 mg/mL) and incubate for 30 min. at 37oC. Later, add the Phenol, Chloroform and Isoamyl alcohol mixture in the ratio (25:24:1) in an equal amount to remove the protein and other moieties from the suspension. Mix the suspension for couple of times by inverting the tubes. Further, centrifuge at 10,000 rpm for 10 min at 4oC. The aqueous layer is transferred to a fresh 1.5 mL pre chilled centrifuge tube and two volumes of isopropanol is added. The contents are mixed by inverting the tubes for 4 to 5 times and incubate the tubes in standing position for 5 min at room temperature. During the incubation the plasmid will precipitate and it is collected in pellet form after centrifuging, the suspension at 10,000 rpm for 10 min at 4oC. Then discard the supernatant and the wash the pellet with 70% ethanol and spin for 1 min at 10,000 rpm to recover the plasmid. Discard the supernatantand collect the pellet at the bottom of the tubes and after its completely drying, dissolve in 25-30 μl of T10E1 (pH 7.4) (Sambrook and Russel, 2001).
Copyright © 2018-2023 BrainKart.com; All Rights Reserved. Developed by Therithal info, Chennai.