Note: All volumes are calculated to cater for four plates
per point.
Base Agar
1. Melt 1% Agar (DNA grade) in microwave, cool to 40°C in a water bath. Warm 2X DMEM/F12 + additives to 40°C 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 4°C for up to
1 week.
1.
Melt 0.7% Agar (DNA grade agarose) in microwave, cool to 40°C in a water bath. (It is important not to exceed 40°C, 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 4°C 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 37°C 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.