...transfected cells. k. Incubate cells at 37°C, 5% CO 2 for 24 hours. Day 4: l. Harvest media from cells and transfer...concentration for your cell line before initiating your experiment. Day 1: a. Plate target cells in ten 6 cm plates...plates from 1-10 and add appropriate puromycin-containing media to cells. Days 3+: e. Examine cells each day...Selection Day 1: a. Plate target cells and incubate at 37°C, 5% CO 2 overnight. Day 2: b. Target cells should...for your target cells. The final concentration of puromycin should be from 1-10 μg/mL in 1 μg/mL increments...of Contents A. pLKO.1-TRC Cloning Vector A.1 The RNAi Consortium A.2 Map of pLKO.1 A.3 Related plasmids...Order oligos compatible with pLKO.1 C. Cloning shRNA oligos into pLKO.1 C.1 Recommended materials C.2 Annealing...