Lysin purification protocol
Pellet 500ml cells (5min x 5000RPM).
Resuspend in 20ml MBBM, add 20µl tetracyline.
Add 150µl purified PBCV incubate 16hrs under fluorescent illumination with a 16 hr light/8 hr dark cycle at 25°C on an orbital shaker (~95 rpm).
Collect cells and virus by centrifugation at 25,000RPM (50.2Ti) for 17 min at 20°C.
Re-suspend cells in 20ml 25mM KOH, 50mM MOPS, 2mM Na2EGTA, 0.1M LiCl(pH 7) (wash).
Centrifuge 25,000RPM for 17 min at 20°C, discard supernatant.
Suspend pellet in 1.5ml 25mM KOH, 50mM MOPS (pH 7), 60µl 10M LiCl, 30µl 0.1M Na2EGTA incubate 50 min at 37°C.
Centrifuge 25,000RPM for 17 min at 20°C.
Collect supernatant (avoid DNA).
