Note: All volumes are calculated to cater for four plates per point. Values in bold represent plating in 6cm dishes.

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 mm dish (2.5mL), 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/35mm plate (10,000/6cm plate), therefore you need 20,000 (40,000)/tube for four plates. Adjust cell count to 200,000 (400,000) 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 (5mL) 2X RPMI + 10% or 20% FCS and 3mL (5mL) 0.7% Agar to tube with cells, mix gently and add 1.5mL (2.5mL) 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.

Method 2.

Solutions

3.3% agar | autoclaved
1.8% agar |
2x DMEM
lx DMEM
FBS

1. Set up water baths to 37°C and 45°C

2. Prewarm 2x DMEM, DMEM and FBS to 37°C

3. Boil 3.3% agar to dissolve. Allow to cool, then place all media and Agar in 45°C water bath for 10 min.

4. In 50 ml tube mix:
3 mL 3.3% agar
5 mL 2x DMEM
10 mL 1x DMEM
2 mL FBS

5. Pipet 2 ml/well into 6 well clusters

6. Allow to set without disturbing in hood for 30 min.

7. Turn water bath to 41°C

8. Boil 1.8% agar to dissolve; allow to cool

9. Transfer 2x DMEM, FBS and agar to 41°C water bath for 10 min.

10. Detach cells and dilute to 3.4 x 10⁵ cells/mL in DMEM

11. In 50 ml tube mix:
3 mL 1.8% agar
6.7 mL 2x DMEM
3.6 mL DMEM
1.7 mL FBS

12. Allow to cool for 20 min then add 2 mL cells

13. Carefully pipet 0.5 mL on top of base layer of agar and allow to set in hood for 30 min. Then transfer to incubator.