sect71
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 waterbath. Warm 2X RPMI + 20% FCS to 40¡C in waterbath. Allow at least 30 minutes for temperature to equilibrate.
2. Mix equal volumes of the two solutions to give 0.5% Agar + 1X RPMI + 10% FCS.
3. Add 1.5ml/ 35 m Petri dish, allow to set. The plates can be stored at 4¡C for up to 1 week.
Top Agar
1. Melt 0.7% Agar (DNA grade agarose) in microwave, cool to 40¡C in a waterbath. (It is important not to exceed 40¡C, otherwise cells will be killed). Also warm 2X RPMI + 20% FCS to the same temperature.
2. Trypsinise cells and count. It is very important to have a positive control line (eg. ras transformed).
3. You require 5,000 cells/plate, therefore you need 20,000/tube. Adjust cell count to 200,000 cells /ml.
4. Add 0.1ml of cell suspension to 10ml yellow capped 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 RPMI + 10% or 20% FCS 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.