Spheroplasts Falcon

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• Prepare 50 ml falcon tubes with 15ml of medium plus antibiotics.
• Transfer 150 uL of a saturated preculture.
• Put falcons in incubator *without lid* but very well fastened with some tape from the top.
• Let the tubes at 37C until it reaches OD600 = 0.6, usually this is 3 or 4 hours.
• Then induce the cells using the appropriate inductor and transfer the tubes at your favorite temperature and time
• Centrifuge the falcons at 3700 rpm during 10min at 4C.
• Discard the supernatant in a bottle and resuspend the pellet with Tris pH 8 @ 20mM, NaCl 250mM, Sucrose 20% and lysozyme 1mg/mL. Use 15ml.
• Resuspend pellets and then Incubate for 30’ on a rocking platform in cold room.
• Centrifuge falcons 3700 rpm during 10min at 4C. Pour supernatant out
• TAKE CARE TO NOT LEAVE ANY TRACE OF SUCROSE !!!!!
• Incubate the plates in freezer for at least 20 minutes
• Use your favorite Lysis buffer (inlcude DNAse) and resuspend in 2.5 -3 ml per tube.
• Resuspend pellets. If resuspension was not efficient use a pipet to help resuspending.
• Finally incubate for 20’ on a rocking platform in cold room. Then leave plates on ice.

You now have your lysate. You can use it to run a gel with the total protein or spin it to collect the soluble fraction.

This protocol is for the moment untested.

Tassos 14:33, 6 December 2007 (CET)