Yeast glass beads extract protocol

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COHscientist
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Yeast glass beads extract protocol

1) Wash the pellet with 1mL cold d2W
2) Wash with 500uL of Buffer A spin down for 1min max speed
3) Resuspend with 200ul of Buffer A and add glass beads at the same level of liquid
4) Vortex for 1 min and put it on ice for 1 min, repeat 5 times
5) Spin down the pellet for 1min with max speed
6) Take supernatant to 1.5mL tube
7) Add 25uL 2M KCl
8) Incubate it on ice for 5 min
9) Centrifuge for 20 min at max speed
10) Take supernatant to new tube

Buffer A
10mM Hepes pH7.8
1.5mM MgCl2
10mM KCl
0.5mM EGTA
0.1 mM 2-mercaptoethanol

add protease inhibitor to Buffer A
1mM PMSF
1mM Benzamidine
1uM leupeptin
1uM Pepstatin