-Galactosidase assays 1. Thaw the system components and mix each component well before use. Place the Assay 2X Buffer on ice. 2. Pipet 150 l of the diluted cell extracts (100 l of cell extracts + 50 l of 1X Reporter Lysis Buffer) into labeled tubes 3. Add 150 l of Assay 2X Buffer to each of the tubes. 4. Mix all samples by vortexing briefly. 5. Incubate the reactions at 37 C for 30 min. [Color development continues for 3 h] 6. Stop the reactions by adding 500 l of 1 M Sodium Carbonate. Mix by vorexing briefly. 7. Read the absorbance at 420 nm. * Standard Curves -Galactosidase Volume of Volume of 1X Standard (milliunits) 1:10,000 Stock (l) Reporter Lysis Buffer (l) 0 0 150 1.0 10 140 2.0 20 130 3.0 30 120 4.0 40 110 50 100 60 90 1:10,000 Stock : Add 10 l of 1u/l -galactosidase to 990 l of 1X Reporter Lysis Buffer and mix. Then add 10 l of this 1:100 dilution to 990 l of 1X Reporter Lysis Buffer and mix it to make a 1:10,000 stock solution. (1) Follow the protocol described above, Steps 3-7. (2) Plot the absorbance at 420 nm versus concentration of –Galactosidase standards. 1 Dr. Lee’s Lab