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- Grow 5 ml of yeast culture to saturation (1 or 2 days) in YPD (rich media)
- Collect cells using a falcon tube in a clinical centrifuge at 3/4 speed around 5 min.
- Pour off supernatant and resuspend cells in 500 ul water and transfer to a microfuge tube.
Spin in microfuge 10 seconds full speed and pour off water. Use residual water left behind
to resuspend the pellet.
- To pellets add 200 ul Winston juice, around .3 g (2 bead spoonfuls) acid washed glass beads,
and 200 ul phenol/chloroform.
- Vortex at full speed 5 minutes.
- Add 200 ul TE (10mM Tris, 1mM EDTA) to the preps and microfuge on high 5 min.
- Transfer supernatant to new tubes, add 200 ul phenol/chloroform, and vortex again for 3 minutes.
Spin for a few sec.
- Transfer supernatant to new tube, add 1 ml 100% EtOH, invert to mix, and microfuge on high 5 min.
Discard supernatant and last remnants of EtOH. Let pellets air dry a few min.
- Resuspend in 400 ul TE, add about 20 ug RnaseA, and incubate at 37°C
for 5 minutes. (The pellet is hard to resuspend so don't work too hard. It's much easier after the RnaseA digestion).
Winston Solution
2% Triton X-100
1% SDS
100mM NaCl
10mM Tris pH8
1mM EDTA
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