Haynes Lab:Notebook/CRISPR Editing/2015/08/25

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08/25/2015 Transfect gal4+puro and gal4+dox with g034, collect at 24, 48, and 72 hours Step 1: Making .5ug/10ul plasmid stocks ' 1 well 19 wells ug g034 0.5 9.50 ul g034 ul water Step 2: make DNA + optimem mixes ' 1 well 19 x g034 39 add 741ul optimem Step 3: add plus reagent to DNA+ opti ' 1 well g034 add 19ul plus reagent step 4: Make optimem + lipo mastermix ' 1 well for 18 wells (x 19.2) optimem 47 902.4 lipo 3 57.6 Step 5: mix dna mix and lipo mix ' 1 well x 19 g034 50 add 950 lipo mix Step 6: add lipo mix to cells add 100ul of mix to each well Transfected siRNAs EZH2 and SUZ12 into gal4-EEDs with dox Cells plated yesterday looked good. Cells plated at 30% were at about 80% Cells plated at 10% and 20% were at about 50% Transfected 2 wells for each siRNA and each cell type For siRNA complexes ' per well 3.1 20uM siRNA duplex 2.5 7.75 Optimem 46 142.6 Oligofectamine 1.5 4.65 Total 50 155 For blank transfections ' per well 3.1 water 2.5 7.75 Optimem 46 142.6 Oligofectamine 1.5 4.65 Total 50 155 Mix optimem and siRNA or water 7.75ul each siRNA + 124ul Optimem in 1.5mL tube 7.75ul water + 124ul Optimem in 2 1.5mL tubes Mix oligofectamine and optimem 19.8ul oligofectamine + 79.2ul optimem let stand for 10 minutes Mix DNA and oligofectamine add 23.25ul oligofectamine to tubes with RNA or water let stand for 30 minutes While complexes form, prep cells Removed media Wash in 100ul PBS Aspirate PBS Add 200ul of plain DMEM Add complexes to cells Add 50ul complexes dropwise to each well After 4 hours, add 125ul DMEM + 3x serum to each well.