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Revision as of 10:57, 10 February 2008
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10:57, 10 February 2008
New page: ==Spheroplasts lysis starting from Falcon tube cultures== *Prepare 50 ml falcon tubes with 12.5ml of medium plus antibiotics. *Transfer 125 uL of a saturated preculture. *Put falcons in i...
==Spheroplasts lysis starting from Falcon tube cultures==
*Prepare 50 ml falcon tubes with 12.5ml of medium plus antibiotics.
*Transfer 125 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 about 3 hours.
*Then induce the cells using the appropriate inductor and transfer the tubes at your favorite temperature and time (0.5 mM IPTG and 5 hrs at RT are a good start)
*Centrifuge the falcons at 3700 rpm for 10min at 4C.
*Discard the supernatant in a bottle and resuspend the pellet in 12.5 ml Tris pH 8 @ 20mM, NaCl 250mM, Sucrose 20% and lysozyme 1mg/mL.
*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 (overnight is a good choice at this stage...)
*Use your favorite Lysis buffer (inlcude DNAse) and resuspend in 2.5 per tube (a good buffer is for example 10 mM Tris pH 7.5, 300 mM NaCl; 1 mM MgCl2; DNAse; Inhibitors cocktail w/o EDTA).
*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.
*Transfer a total of 2ml per sample to a 2ml Eppendorf tube
*Spin 10 min at 14k
*Take supernatant, add magnetic His beads, shake for 30 min is Cold room.
*Pool down beads with magnetic rack, remove supernatant
*Wash beads 1x with 1 ml lysis buffer
*Elute proteins in 30 ul lysisbuffer +0.5M Imidazole
*If you get lucky you even have enough not only to run a gel but also to crystallize in Fluidigm chips at this stage
''This protocol has been adapted from the Grenoble one by Tassos, based on Dene's testing in Falcon tubes and Vangelis's magnetic beads preferences and tested successfully by Bas (Piet Borst group) and Vangelis.''
[[User:Tassos|Tassos]] 19:46, 6 December 2007 (CET)
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