PLASMID ISOLATION:

Aim of the experiment: To isolate plasmids viz.pGPTV and pUC-ANNEXIN from E.Coli.

Introduction:
Plasmid isolation involves 4 steps 1) Harvesting. 2) Alkali lysis. 3) Plasmid precipitation. 4) Purification Harvesting: - overnight grown culture was centrifuged to pellet down the cells and pellet was resuspended in STE and again recovered by centrifugation. Alkali lysis: - The pellet was treated with solutions I, II and III whose constituents help to lyse the cells and precipitate the cell debris and protein content by centrifugation. Plasmid precipitation: - The supernatant was treated with 0.6 V of isopropanol in order to selectively precipitate only plasmid DNA. The DNA precipitated was recovered by centrifugation. Purification: - The pellet thus obtained was dissolved in TE and treated with RNaseA, further phenol:chloroform extraction was done to remove protein and cell debris. The purified DNA was precipitated using 0.1 volumes of 3M-sodium acetate and 100% ethanol; finally it was washed with 70% ethanol, dried and stored at -70 oC.

PROTOCOL:
1. 100 l of bacterial glycerol stock was added into 50ml of LB broth containing the respective antibiotics (that is Kanamycin for pGPTV and Ampicillin for pUC Annexin).

2. The conical flasks were incubated overnight at 37 oC on the orbital Shaker. 3. The cultures were transferred into sterile centrifuged tubes and Chilled on an ice bath for 5 minutes at 4 oC. 4 The cells were harvested by centrifugation at 6,000 rpm for 6 minutes at 40C. 5 5 ml of ice-cold STE solution was added and the bacterial pellet was resuspended in it. This helps removing any media in the bacterial pellet. 6 It was then centrifuged at 6,000 rpm for 6 minutes. 7 The supernatant was discarded, and to the pellet, 3 ml of solution-I was added. The vial was vortexed gently to get uniform suspension. 8 The vial was kept in ice for 10 minutes and shifted to room temperature. 9 6 ml (double the volume of Solution I) of freshly prepared solution-II was added to the vial and mixed well. The tubes were incubated for 10 minutes at room temperature and shifted to ice for 1 minute.

10 4.5 ml (average of the volumes of Solutions I and II) of solution-III was added and mixed well and then kept on an ice-bath for 10 minutes. If treated with solution-III for longer periods of time renaturation of chromosomal DNA may also take place bringing about contamination of plasmid DNA. 11 The centrifuge tubes were centrifuged at 12,000 rpm for 12 minutes. 12 The supernatant was transferred to fresh tubes (the cell debris formed a thick white pellet) and 0.6V of isopropanol was added and mixed well. The tubes were left at room temperature for 30 minutes to allow the DNA to precipitate.

13 They were then centrifuged at 10,000 rpm for 10 minutes. The DNA was observed as a white pellet sticking to the sides of the tubes. 14 The tubes were air-dried for few minutes and the pellet was dissolved in 0.8ml of 1X TE. 15 The contents were shifted into 2ml eppendorf tubes and 10-12 l of RNaseA (10mg/ml concentration) was added into each tube. The tubes were then incubated at 37 oC for 30 minutes. This step allows the denaturation of any RNA content in the preparation. 16 After 30 minutes Phenol: Chloroform extraction the supernatant is carried out. An equal volume of phenol:chloroform:isoamyl alcohol (in the ratio of 25:24:1 v/v) was added and centifuged at 12,000 rpm for 12 minutes. The supernatant was transferred to fresh tubes. This process was repeated twice or thrice. 17 Equal volume of chloroform: isoamyl alcohol (24:1 v/v) mixture was added to the supernatant and centrifuged at 12,000 rpm for 12 minutes. supernatant was transferred to fresh eppendorf tubes. 18 To the supernatant 0.1V of 3M sodium acetate was added and mixed gently. Two volumes of absolute alcohol was added and kept at- 20 oC over night. This step allows the re-precipitation of the plasmid DNA. 19 The vial was centrifuged at 12,000 rpm 12 minutes at 4 oC. The pellet contained plasmid DNA. 20 The pellet was washed with ice cold 70% ethanol. 21 The pellet was dried in a speed vacuum dessicator for about 10 minutes. 22 The pellet was re-dissolved in 30 l-40 l of 1 x TE buffer (depending on the size of the pellet). The

RESULT:
The plasmids pUC/ANNEXIN and pGPTV were isolated.

The isolated plasmids were run on agarose gel.