Samples collected from cell growth on Saturday were lysed today using enzymatic cell lysis.
Cell samples were pooled together in one 50 mL Falcon tube, with a final cell weight of 7.5 grams in 22.5 mL of lysis buffer.
Lysozyme was added to thawed cell solution on ice (4.5 mg, or approximately 0.0045g) and incubated on ice, swirling periodically for 30 minutes.
PMSF was added to the cell solution following lysozyme at a final concentration of 1mM from a 100mM stock (225uL of 100 mM PMSF stock).
While stirring vigorously, 11.25g of Deoxycholic acid was stirred into solution. Lysate was then incubated at 37 degrees for 20 minutes.
20 uL of DNase I was added to the lysed samples, and incubated for 30 minutes at RT on a shaker.
Sample centrifuged for 15 minutes at 3,300 xg at 4 degrees. Supernatant transferred to a 50 mL conical at heat treated for 10 minutes at 80 degrees in a water bath. Cells were respond and the supernatant was recollected as the protein extract.