User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/17: Difference between revisions
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== | ==Procedure== | ||
#The tubes containing our cells in Tris buffer (with protease inhibitors) were thawed | |||
#They were placed in the centrifuge for 25 minutes at 3500rpm | |||
#The pellets were then sonified | |||
##30 seconds sonification followed by 30 seconds on ice until the pellet was loose and liquid | |||
#Once liquefied, the black supernatant was put back in the tubes and the solution was centrifuged at 20,000rpm four 3 hours at 4C | |||
##The tubes were spun for an additional 30 minutes under the same conditions in order to prepare the materials for the next step | |||
#A saturated solution of Ammonium Sulfate was prepared by adding Ammonium Sulfate powder to 10mL of distilled H2O until it would no longer go into solution | |||
##Amount added was the equivalent of 5mL of powder | |||
#The tubes were collected from the centrifuge and the supernatant was placed in a 50mL falcon tube | |||
##Both the supernatant and the saturated ammonium sulfate solutions were placed on ice for 10 minutes | |||
#A volume of saturated ammonium sulfate solution equivalent to 1/4 of that of the supernatant was added to the latter | |||
##in this case, 7.5mL of the saturated solution were added to the 30mL of supernatant | |||
##Notice solution become cloudy | |||
#This mixture was left on ice for 30 minutes and then placed in the centrifuge at 4C, 10000g for 30 minutes | |||
##The supernatant was placed in dialysis tubing and that was placed in 4L of neutral phosphate buffer overnight | |||
##The pellet was resuspended in 50mM Tris and placed in the fridge | |||
Revision as of 07:33, 18 July 2014
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Procedure
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