DNA EXTRACTION PROCEDURE (CsCl) - RHIZOBIUM

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. Re-pellet 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 - 37C until cell lysis (about 30 min - 1 hr).
6. Extract 3X with equal volume of Tris-equilibrated phenol and centrifuge at 7,000X rpm for 15 min. Transfer top layer to new tube (avoid interface). Re-do this step if necessary.
7. Extract 2X with diethyl ether. Remove and properly discard top layer after centrifugation at 5,000X rpm for 5 min. Evaporate ether overnight in fume hood, or heat in 68C 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 10,000X g for 5 min. Transfer top layer to a new tube.
10. Place in Quick-seal tubes and centrifuge in Ti50 rotor at 36,000 rpm for 48 hr.