S. pombe Cell Lysis with Krups Coffee Grinder Method
a. Culture cells on YES or EMM plate (if selection of plasmid required) for 3 days, Inoculate a 10 ml culture and grow overnight, use this to inoculate a 50ml culture and grow O/N. Use the 50ml to inoculate a 3litre culture. Grow 3 litres of cells overnight to OD 0.8-1
b. Spin cells at 5000 rpm for 20 minutes and aspirate media
c. Resuspend cells in STOP buffer. Spin down pellet and snap freeze in liquid nitrogen (Pellet size should be about 8 ml)
Equipment: Coffee grinder (Krups 203-42, $19,99 from Linen N’ Things or other retailer)
d. Transfer cell pellet into coffee grinder (Prechill coffee grinder cup and coffee grinder lid with dry ice), add to pellet approx. 20g of dry ice.
e. Grind continuously for 3-5 mins. (Very Important: Be sure to keep the coffee grinder lid chilled with dry ice during the grinding, by placing a block or bag of dry ice on it. This prevents the powered cells from sticking to the lid and heating up)
f. Cells should be ground to a fine powder. You can check under the microscope. You can expect between 50-60% cell breakage.
g. Resuspend powdered cells in IP lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 0.5% TX100) and protease inhibitors (PMSF and protease inhibitor cocktail tablets (Roche). Try to keep the volume low at this point – you can always add more later. I usually add about 15 ml for a 20 ml pellet to start.
h. Keep lysate at 4ºC during thawing (it can take up to about 15 minutes to thaw depending on how much dry ice was added during grinding)
j. Lysate should be put into pre-chilled plastic centrifuge tube and spun for 30 minutes @ 20K rpm.