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Protocol

CLONING IN SOFT AGAR
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Media Stock Solutions (for 100 mL) Stock A Stock B Stock C Stock D Stock E (10% serum) (5% serum) (2.5% serum) (2% serum) (1% serum) l0x RPMI 10 mL 10 mL 10 mL 10 mL 10 mL serum 10 mL 5 mL 2.5 mL 2 mL 1 mL NaHCO3 (7.5%) 2.6 mL 2.6 mL 2.6 mL 2.6 mL 2.6 mL H20 55.9 mL 60.9 mL 63.4 mL 63.9 mL 64.9 mL glutamine 1 mL 1 mL 1 mL 1 mL 1 mL pen/strep (200x) 0.5 mL 0.5 mL 0.5 mL 0.5 mL 0.5 mL
Underlay 1. Pipette 80 mL of stock solution media (see following page) into sterile 125 mL bottle. Place in 48°C heating block in hood for 5-10 minutes. 2. Add 20 mL sterile 2.5% agarose (made up in ddH20 and autoclaved); mix by pipetting up and down. Make sure agarose is not too hot. I usually let it sit for 5-10 minutes at 60°C after boiling. 3. Pipette agarose/media solution into 6-well plates, using 4 mL/plate. Make sure there are no bubbles on surf ace of agar. Let harden. At this point, the plates can be wrapped and stored at 4°C overnight until ready to use. Before use, place in 37°C C02 incubator for at least 1 hour. Overlay 1 . Pipette 80 mL of stock solution into sterile 125 mL bottle. Place in heating block at 48°C. 2. Add 100 mL sterile H20 + 10 mL sterile 2.5% agarose and mix by pipetting up and down. 3. To a sterile capped tube (not conical), add 2 mL agarose/media solution and desired number of cells to plate. (Prepare cells by trypsin-treating the monolayer, pelletting cells,washing once with serum-free medium, and resuspending in medium supplemented with desired percentage of serum. Count cells and make appropriate dilutions to obtain the desired amount of cells in a volume no greater than 200 mL). 4. Vortex tube and quickly pour over underlay. Let harden, and incubate at 37°C, 5% CO2.
CLONING IN SOFT AGAR - IN STERILE HOOD

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