DNA Extraction of Rhizobium (CsCl Method)

1. Grow starter culture overnight in 5 mL GYPC medium.
2. Inoculate 200 mL GYPC and grow cells to mid-late log phase (visual).
3. Pellet cells by centrifugation. Resuspend cells in 5 mL 10 mM Tris (pH 8.0), 4 mM EDTA. Then add 150 mL more buffer to wash cells.
4. Repellet cells and resuspend in 4 mL 50 mM Tris (pH 8), 20 mM EDTA in a small flask. Place on ice for 15 min.
5. Add 0.4 mL proteinase K (2 mg/mL, fresh); swirl for 5 min. Then add 0.1–0.2 mL sarcosyl (10%). Swirl gently at 30°C–37°C until cell lysis (about 30 min–1 hr).
6. Extract 3X with equal volume of Trisequilibrated phenol and centrifuge at 7000X rpm for 15 min. Transfer top layer to new tube (avoid interface). Redo this step if necessary.
7. Extract 2X with diethyl ether. Remove and properly discard top layer after centrifugation at 5000X rpm for 5 min. Evaporate ether overnight in fume hood, or heat in 68°C water bath for 30 min.
8. Adjust aqueous phase to 8.7 mL with buffer. To this, add 8.3 g CsCl and 0.9 mL ethidium bromide (10 mg/mL).
9. Extract with equal volume chloroform (mix by inverting) and centrifuge at 10000X g for 5 min. Transfer top layer to a new tube.
10. Place in Quick-seal tubes and centrifuge in Ti50 rotor at 36000 rpm for 48 hrs.