YEAST HISTONE PREP (Modified, 2/17/2003)

General protocol for releasing plasmids from yeast so they can be sub-sequentially used to transform bacteria.

1. Grow a 5 mL culture in YPD overnight.

2. Collect the culture in a 1.5 mL Eppendorf tube.

3. Resuspend the cells in 200 ul of lysis buffer (10 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 2% Triton X-100 and 1% SDS) and add 200 ul of phenol:chloroform:isoamyl alcohol (25:24:1) and 0.3 g (roughly 0.3 ml) of 0.5 mm glass beads.

4. Vortex the tube at top speed for 2 minutes.

5. Add 200 ul of TE and vortex again for a few seconds.

6. Spin the tubes for 5 minutes at RT at top speed in an Eppendorf centrifuge.

7. Transfer the aqueous (upper) phase (380 ul) to a fresh Eppendorf tube, using a new pipette tip for each sample. Discard the tube with the glass beads.

8. Add 2 volumes of 100% ethanol and mix thoroughly.

9. Centrifuge the Eppendorf tube 5 minutes at RT.

10. Discard the supernatant taking care not to dislodge the pellet.

11. Wash the pellet with 0.5 ml of 70% ethanol.

12. Air-dry the DNA pellet for 10 minutes.

13. Resuspend in 25 uL of TE. Use 10 uL for transformation of 200 uL of bacteria.