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Note: All volumes are calculated to cater for four plates per point.


Base Agar


1. Melt 1% Agar (DNA grade) in microwave, cool to 40C in a water bath. Warm 2X DMEM/F12 + additives to 40C in water bath. Allow at least 30 minutes for temperature to equilibrate.


2. Mix equal volumes of the two solutions to give 0.5% Agar + 1X DMEM/F12 + additives.


3. Add 1.5ml/ 35 m Petri dish, allow to set. The plates can be stored at 4C for up to 1 week.


Top Agar


1.  Melt 0.7% Agar (DNA grade agarose) in microwave, cool to 40C in a water bath. (It is important not to exceed 40C, otherwise cells will be killed). Also warm 2X DMEM/F12 + additives to the same temperature.


2. Trypsinize cells and count. It is very important to have a positive control line.


2.  You require 5,000 cells/ plate, therefore you need 20,000/tube. Adjust cell count to 200,000 cells /ml.


3.  Add 0.1ml of cell suspension to 10ml centrifuge tubes.


5. Label 35mm Petri dishes with base agar appropriately (it is a good idea to remove the plates from 4C about 30 minutes prior to plating to allow them to warm up to room temperature).


6. For plating add 3ml 2X DMEM/F12 +Additives and 3ml 0.7% Agar to a tube, mix gently and add 1.5ml to each replicate plate (usually plate out in triplicate). NOTE: Only do one tube at a time so that agar does not set prematurely.


7. Incubate assay at 37C in humidified incubator for 10 - 14 days.


8. Stain plates with 0.5ml of 0.005% Crystal Violet for >1 hour, count colonies using a dissecting microscope.