DNA Isolation from Rhizobium by Phenol Chloroform Method

 

1. Grow Rhizobium cells in 5 ml of YEMB at 200 rpm till the O.D (600 nm) reaches 0.6-0.8.
2. Pellet the cells by centrifugation at 10,000 rpm for 15 mins.
3. Wash the pellet with TE buffer (10T/1E).
4. Dissolve the pellet in 400 μl TE.
5. Add 40 μl 10% SDS and 5μl Proteinase K (20mg/ml).
6. Incubate at 56 ^o C for 45 mins.
7. Add 400 μl of tris saturated phenol (pH 8.0).
8. Centrifuge at 10,000 rpm for 10 mins.
9. Take the supernatant and add 200 μl of tris saturated phenol and 200 μl of chloroform: isoamyl alcohol (24:1).
10. Again take the supernatant and repeat the above step.
11. Take the supernatant and add 400 μl of chloroform: isoamyl alcohol (24:1).
12. Repeat the above step.
13. Take the supernatant and add 0.1 volume of 3M sodium acetate and 2 volume of chilled absolute ethanol.
14. Incubate at -20 ^o C for 2 hrs.
15. Centrifuge at 15,000 rpm for 30 mins in a cooling centrifuge at 10-15 ^o C.
16. Decant the supernatant and wash the pellet with 70% ethanol by centrifugation at 10,000 rpm for 15 mins.
17. Decant the supernatant and dry the pellet at room temperature.
18. Suspend the pellet in 50 μl of TE.

 

Reagents
 

Composition of YEMB (Yeast Extract Mannitol Broth)
Mannitol	10.0 g
K2HPO4	0.5 g
Yeast extract	0.5 g
MgSO4.7H2O	0.2 g
NaCl	0.1 g
Distilled water	1 litre
pH	6.8