User:Daniel A. Vargas/Notebook/General lab notebook/2015/10/15

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Passaging and Plating of Trial 3

Using a 90% confluent T-75 Flask of 2 day old thawed cells, new plates were passaged.

1. Media was removed from plate followed by adding PBS wash

2. PBS was removed and tripsin efta was added for 2 min

3. 8mL of DMEM was added to inactivate trypsin

4. the 10mL was then added to a 15mL falcon tube and spun at 1000rpm for 2min

5. Supernatant was removed leaving the cell pellet

6. 10mL of DMEM 10%FBS 1% P/S was used to break up pellet

7.five 10cm plates were made with 1mL of cells added to each.

8. Plates were brought up to 10mL with their required antibiotic

9.10uL of 1mg/mL of Dox was added to the 192 hour plate