Team:UCLA/Notebook/Recombinant Expression/18 July 2015

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Suspension of Cells in Lysis Buffer

The flask is removed from the shaking incubator and the contents are divided into two 500mL containers. The containers are weighed and a few drops of water is added to the lighter one to ensure proper counterbalance in the centrifuge. The two containers are centrifuged at 5,000g at 4 degrees Celsius for 15 minutes. The supernatant is decanted and the cell pellets are weighed, and then suspended in 1X Lysis Buffer in a 1 to 3 weight of cell pellet to volume of buffer, respectively, ratio.

Weight of Empty 500mL container: 80.56g

Weight of 1st 500mL container with Cell Pellet: 84.06g

Weight of 2nd 500mL container with Cell Pellet: 84.55g

Weight of 1st Cell Pellet: 84.06g-80.56g = 3.5 g

Weight of 2nd Cell Pellet: 84.55g-80.56g = 3.99g, round to 4 g

Volume of Buffer Needed:

3.5 x 3 = 10.5 mL

4.0 x 3 = 12.0 mL