Haynes Lab:Notebook/CRISPR Editing/2015/06/30

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06/30/2015 transfected Luc14s gal4-eeds with dox with 2 dilutions of g034 in triplicate. Step 1: Diluting the DNA ' ug of DNA for 3.2 wells ul plasmid water (32ul total) g034 0.1 0.32 g034 0.01 0.032 g034 0.1 0.32 g034 0.01 0.032 Step 2: make DNA + optimem mixes ' 1 well 3.2 x g034 0.1 39 add 124.8ul optimem g034 0.01 39 add 124.8ul optimem g034 0.1 39 add 124.8ul optimem g034 0.1 39 add 124.8ul optimem Step 3: add plus reagent to DNA+ opti ' 1 well 3.2 x g034 0.1 1 add 3.2ul plus reagent g034 0.01 1 add 3.2ul plus reagent g034 0.1 1 add 3.2ul plus reagent g034 0.01 1 add 3.2ul plus reagent Step 4: Make optimem + lipo mastermix ' 1 well for 12 wells (x 13.2) optimem 47 620.4 lipo 3 39.6 Step 5: mix dna mix and lipo mix g034 0.1 add 160 lipo mix g034 0.01 add 160 lipo mix g034 0.1 add 160 lipo mix g034 0.01 add 160 lipo mix Step 6: add 100ul to each well Luciferase assay on Luc14s, Gal4-EEDs+puro, and Gal4-EEDs+dox (6/22 and 6/26) Collected transfected gal4-eed cells with dox or puro for flow and gDNA extraction: aspirate media add 400ul PBS, rock plate, aspirate PBS add 200ul trypsin, let sit for a few minutes, tap plate to unstick cells add 500ul media, pipette to collect cells, move to 1.5mL microcentrifuge tube remove tubes from hood spin in benchtop centrifuge at 0.2x1000RCF for 3 minutes remove media with P1000 resuspend in 400ul cold PBS filter 200ul for flow, use remaining 200ul for gDNA extraction Plating cells for transfection with siRNAs with oligofectamine oligofectamine calls for no antibiotics, serum-free media, and for cells to be 30-50% confluent at time of transfection trypsinized Gal4-EED cells that had been induced with dox at 3PM on 6/26 filtered some DMEM only for serum-free media Plated 0.1x10^6 cells per well of a 6-well plate