01:694:215 Lecture Notes - Lecture 3: Ura3, Spectrophotometry, Sirtuin 1
Document Summary
Null mutant sir2 fail to repress lacz expressed blue. Wild type sir2 repress no lacz expressed white. Transfer cells to filter freeze cells in liquid n break open cell and proteins thaw cells on buffer with xgal cells lyse releasing lacz. Get a window time- eventually all assays will turn blue. Dilute an aliquot 20x; take od600 to count cells. Mix 250 ul cells with x buffer, sds, chcl3 to lyse cells. Measure how much lacz is expressed by how yellow it is. Normalize for the amount of cells that you put into each reaction. Time- how long did you let the reaction go. X units = 1000 x od420 / od6000 x dilution factor x volume x time. Od420- amount of color measured at the end of the reaction (the higher, the more cells) Od600- optical density of the cell suspension added to the reaction; how many cells there are in a given volume of the cell suspension.