DNA ISOLATION FROM BACTERIA
Reagents:
1. Solution I: 5 ml of 1M glucose, 2.5 ml of 1M Tris (pH-8), 2ml of 0.5M EDTA are mixed and the volume was made up to 100 ml with distilled water.
2. Solution II: Mix 1 ml of 10% SDS and 0.5ml of 4N NaOH and make the volume upto 100ml using distilled water.
3. Solution III: 60 ml of 5M Potassium acetate and 11.5 ml of glacial acetic acid and should be mixed and the volume was made upto 100ml with distilled water.
4. TE Buffer:1ml of 1M Tris should be mixed with 200 µl
Procedure
1. 1µl of overnight grown culture was taken in an appendoff and centrifused at 5000 rpm for 5 min.
2. Discard the supernatant and to the pellet 100 µlof solution-I is to be added and suspent the cells by vortexing
3. Then add 200 µl solution-II and gently tap the appendoff then 150 µl of solution-III to be added. Invert the tube gently 3 to 4 times. Store these appendoff for 5 min at 4 oC and then centrifuge the appendoff at 5000rpm for 5 min
4. Discard the pellet and take the supernatant in a fresh appendoff and add equal volume of chilled Isopropnol
5. Mix well and keep in the cold conditions for 30 min.
6. After incubation centrifuse at 5000rpm for 10 min
7. Discard the supernatant and to the pellet ad 100 µl of 70% ethanol
8. Centrifuse at 5000rpm for 5 min and discard the supernatant and keep the tubes in room temperature for 10 min to remove the trays of ethanol
9. To the pellet add 100 µl of TE Buffer and store it at 4oC..
