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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 lx 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 l05 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.
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