sentence: The cells were grown for an additional 2 h ( A ∼ 1.9 ) , separated into four batches of 500 mL , and harvested by centrifugation at 7000g for 15 min at 4 ° C. Cells were washed twice with 50 mL of buffer A [ 100 mM Tris , 150 mM NaCl , and 5 % glycerol ( pH 7.6 ) ] , flash-frozen in liquid nitrogen , and stored at – 80 ° C. Typically , 3.5 g of cell paste per liter of culture was obtained .