Lysin assay

Lysin assay

Harvest NC64a cells from mid-log culture

Centrifuge 5 min at 3.3K

Wash 3x with sterile BBM (1x with 50mM K-MOPS pH 7, 1M LiCl)

Extract cells w/3ml MeOH and measure fluorescence (delete)

Fluorescence is proportional to cell number

Suspend 5x10exp7 cells in 500ul add lysin, incubate 1hr at 25C (100ul, add Triton to 1%, 5-15 min at 37)

Add 200ul 10% SDS then add 2.5ml BBM, vortex and incubate 30 min at 25C (delete)

Remove cells by centrifuging 2 min at 3.3K (30sec at 5K)

Measure fluorescence of supernatant (Photograph supernatant)