• Grow up an 8 Liter pellet, then centrifuge down to a single pellet in one bottle
• Bring volume up to 250ml with 10mM Tris pH 7.4 and mix well
• Centrifuge and save pellet, discard super.
• Bring volume up to 250ml with 50mM Tris pH 7.4, 0.1% TritonX100, 1mM PMSF
• Mix well, you may need to use a Polytron if it is very thick
• Transfer to a glass beaker and while stirring add drop by drop:
• 25mg freshly made lysozyme (have it at 10mg/ml in water=2.5ml solution)
• Keep stirring until it gets sticky, then continue stirring for 3-4 hours at room temp or 
• overnight in the cold room at 4°
• Sonicate on #10 setting in multiple 2 minute bursts on ice until it gets less sticky
• Centrifuge at 14,000 for 20-30 minutes, save super and discard pellet
• Measure volume of super and add 15.4g/100ml Ammonium Sulfate (30%)
• Stir to dissolve and check pH, do not let pH drop below 7.0 (pH 7.5 is ideal)
• Centrifuge at 14,000 for 20 minutes and save super and discard pellet
• Measure volume of super and add 22.7g/100ml Ammonium Sulfate (50%)
• Centrifuge at 14,000 for 20 minutes, save pellet and discard super this time
• Dissolve pellet in 25-30ml CM buffer
• Dialyze this in 1 Liter CM buffer, changing 2-3 times
• Run over a 40 gram CM column at 1ml/minute

CM Buffer

• 6M Urea
• 25mM MES
• 1mM EGTA
• 15mM BME
• pH 6

Elute using 0-0.3 M NaCl (in CM Buffer). Eluted at around 0.2 M NaCl.