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===II. Sonicate (mixonix sonicator 3000)=== *1. Make HEGX (HEGX: Hepes-KOH (pH 7.2), NaCl, EDTA, Glycerol, TX-100, Complete) *2. Resuspend pellet in 80 mL HEGX @ 0-4C, with complete protease inhibitors. '''added 8 roche complete protease inhibitor tablets (1 per 10 mL)''' *3. Sonicate in HEGX in steel container 10-12 cycles of 45-50 bursts (0.5 seconds ON, 0.5 seconds OFF, (50% duty cycle)). ~15 minutes sonicating total. Temp should not go over 10C, keep consistently on salt ice bath at <4C, or cool on salt bath to -2C to -3C in between cycles. Immerse 1.5X tip diameter into solution, use 1/2 inch tip. Monitor temp with temperature probe with instantaneous feedback. Check transparency to see cell lysis after every few cycles. protein precipitates with over sonication. Solution turns translucent and less murky. *4. Pellet 15K rpm, 30min 4C (BIG OLβ CENTRIFUGE) *5. Keep supernatant and discard pellet
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