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lysis buffer (LB):
20mM Tris, pH8
The idea is to break open cells without any detergents, and to take advantage of
different sedimentation properties of membrane fractions and soluble fractions.
now you have [C] [big pellet] [S2] [P2]
- Take 8-10 OD of cells and resuspend in 200ul LB+PI in a 2ml eppendorf. LB should be ICE COLD.
- Add an equal volume of glass beads.
- ON ICE (in cold room) vortex 6x 1 minute at full speed. Place on ice for
30 seconds between each 1 minute burst.
- Withdraw resulting lysate, place in 1.5ml eppendorf ON ICE.
- Wash glass beads with 2x 100 ul of cold LB+PI. Collect each wash in the same
eppendorf from step #4. The resulting 300ul suspension is your crude lysate [C].
Remove 25ul of [C] and place in 25ul USB.
- Spin the crude lysate for 5 seconds in a COLD EPPENDORF CENTRIFUGE. You will get a big pellet [BP].
- Place supernatant in new eppendorf. Add 275ul LB+PI to the resulting big pellet.
- Redo step #6 on the supernatant. If there is a pellet, remove the supernatant to another tube.
- Now spin the supernatant that is free of large debris for 30 minutes in a COLD EPPENDORF CENTRIFUGE.
- Separate resulting supernatant. This is [S2].
- Resuspend the resulting pellet in 275ul lysis buffer+PI. This is [P2].