based on M.D. Rose, F. Winston, and P. Hieter (1990) Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
|1.||To cell pellet in Eppendorf tube, add 0.3 g (roughly 0.3 ml) of glass beads, 0.2 ml of lysis buffer and 0.2 ml of a 1:1 mix of phenol and chloroform.|
|2.||Vortex the tube at top speed for 2 min.|
|3.||Add 0.2 ml of TE (10 mM Tris, 1 mM EDTA, pH 8.0) and vortex again for a few seconds.|
|4.||Spin the tubes for 5 min (room temperature) at top speed in an Eppendorf centrifuge.|
|5.||Transfer the aqueous (upper) phase (0.38 ml) to a fresh Eppendorf tube, using a new pipette tip for each sample. Discard the tube with the glass beads.|
|6.||Add 2 volumes of 100% ethanol at room temperature. Mix thoroughly.|
|7.||Centrifuge in Eppendorf for 2-3 min at room temperature.|
|8.||Discard the supernatant (use the aspirator; take care not to dislodge the pellet).|
|9.||Rinse the pellet with 0.5 ml of cold, 70% ethanol add the ethanol slowly down the side of the tube, then centrifuge for 3-5 sec.|
|10.||Remove the supernatant. Leave the tubes open and inverted for the pellets to dry. (Or dry the pellets under vacuum.)|
Use 425-600 micron beads. Sigma G-9268 works well for us. To prepare the beads:
- Pour the beads (1 kg bottle) into a 2 litre beaker.
- Fill the beaker with concentrated HCl until the beads are fully submerged. Let stand in a fume hood for 15 min.
- Wash with tap-distilled water until the pH is neutral. It helps to run the water through glass tubing jammed to the bottom of the beaker so that the water flows up from the bottom of the beaker.
- Transfer the beads to a baking dish and bake (overnight is good).
- Pour the beads into a sterile bottle.
- 10 mM Tris, pH 8.0
- 1 mM EDTA
- 100 mM NaCl
- 1% SDS
- 2% Triton X-100
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