Sack: Plating Subclones for Ephys

Sack and Yarov-Yarovoy Labs

Department of Physiology and Membrane Biology
University of California, Davis

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Plating subclones for electrophysiological testing For every 12 colonies picked, you will need: 15 ml appropriate selecting media 24 ml media + 1 ug/ml tetracycline 1 TC-treated 12-well plate 12 TC-treated 35 mm dishes 1 25 ml serological pipet serological pipetor 15 200 ul pipet tips (3 rows of 12) 200 ul pipetor waste box for pipet tips pen 1. Choose 12 wells on the 96-well plate that contain one colony. Choose from the lowest density plate first. 2. Remove media from selected wells, rinse once with 200ul PBS, then add 50 ul trypsin. Watch to see when cells detach. 3. While cells are trypsinizing: put 1 ml media in each well of 12-well plate put 2 ml media with tetracycline in a 35 mm tissue culture treated dish mark each well and dish with name of a chosen well from the 96 well plate 4. Once cells detach, to each well add 200ul of media triturate 5x to suspend cells and remove 200ul suspension plate one drop of suspension in appropriately marked 35mm tissue culture dish. place rest of cells in appropriate well of 12 well plate 5. Add 200 ul media to picked wells of 96-well plate, this makes a backup from straggling cells. 6. Place cells in incubator, patch clamp cells in 35 mm dishes 1-2 days later