Inoculate 1.5L of TB + 0.2% glucose with 100ml saturated culture of MRE 600 cells.
Grow to OD600 of 1.0 at 30ºC.
Pellet cells (5000 rpm, 15 minutes), and wash 1X with ddH2O.
Resuspend in buffer A at 0.5g/ml
Pass suspension through French pressure cell 3X at 4000psi
Centrifuge 30 min. at 40 00xg (19 000 rpm in 50.2).
Dilute supernated 10- fold in buffer B.
Centrifuge 2 hours at 150 000xg (37 000 rpm in Ti 50.2).
Resuspend pellet in 18 mL of buffer B (Note: use Dounce homogenizer with type A pestle).
Incubate vernight with rotation at 4ºC.
Centrifuge 2 hours at 150 000xg.
Resuspend in 20 mL of buffer C.
Centrifuge 2 hours at 150 000xg. Note: Pellet is almost clean, and has approximate consistency of
Resuspend pellet in 500ml buffer C.
Note: Volume of mfrs required must be determined, but should be optimal in this case at about
per 10ml reaction.