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lysis buffer (LB):
20mM Tris, pH8
10mM EDTA
.1M NaCl
.3M sorbitol
The idea is to break open cells without any detergents, and to take advantage of
different sedimentation properties of membrane fractions and soluble fractions.
- 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].
now you have [C] [big pellet] [S2] [P2]
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