DNA Isolation from Rhizobium by Phenol Chloroform Method

1. GrowRhizobiumcellsin5mlofYEMBat200rpmtilltheO.D(600nm)reaches0.6-0.8.
2. Pelletthecellsbycentrifugationat10,000rpmfor15mins.
3. WashthepelletwithTEbuffer(10T/1E).
4. Dissolvethepelletin400μlTE.
5. Add40μl10%SDSand5μlProteinaseK(20mg/ml).
6. Incubateat56^oCfor45mins.
7. Add400μloftrissaturatedphenol(pH8.0).
8. Centrifugeat10,000rpmfor10mins.
9. Takethesupernatantandadd200μloftrissaturatedphenoland200μlofchloroform:isoamylalcohol(24:1).
10. Againtakethesupernatantandrepeattheabovestep.
11. Takethesupernatantandadd400μlofchloroform:isoamylalcohol(24:1).
12. Repeattheabovestep.
13. Takethesupernatantandadd0.1volumeof3Msodiumacetateand2volumeofchilledabsoluteethanol.
14. Incubateat-20^oCfor2hrs.
15. Centrifugeat15,000rpmfor30minsinacoolingcentrifugeat10-15^oC.
16. Decantthesupernatantandwashthepelletwith70%ethanolbycentrifugationat10,000rpmfor15mins.
17. Decantthesupernatantanddrythepelletatroomtemperature.
18. SUSPendthepelletin50μlofTE.

Reagents

CompositionofYEMB(YeastExtractMannitolBroth)
Mannitol	10.0g
K2HPO4	0.5g
Yeastextract	0.5g
MgSO4.7H2O	0.2g
NaCl	0.1g
Distilledwater	1litre
pH	6.8