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 ) .
