- Make 0.6% media-agar mix for the bottom layer.
To make 0.6% agar mix the following components (this makes 200 ml):
- 2X DME 100 ml
- IFS 20 ml
- Pen/Strep 2 ml ddH2O
- 28 ml
- warm components to 37°C
- Add 12.4 g of Noble agar to 100 ml ddH2O and microwave to melt (do not over microwave or you will lose too much volume). Microwave just long enough to completely melt the agar.
- Add 50 ml of agar to the prewarmed mixture that you just prepared.
- swirl to mix avoiding air bubbles.
- add 4 ml of this mix to each 6 cm2 plate
- allow to completely cool while you prepare your cells.
- Prepare the top layer mixture with all of the listed components EXCEPT the Noble agar.
You will need one 50 ml conical for every two cell lines that you are testing. Prepare the following mix and place tube(s) at 37°C to prewarm.
Mix:
- 2X DME 12.5 ml
- IFS 2.5 ml
- Pen/Strep 0.25 ml
- ddH2O 3.5 ml
- Label tubes. For each cell line there should be 3-15 ml conical tubes labeled with 105, 104, or 103 and each of these should have the cell name on the tube.
- Lift two cell lines and pass through a 100