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1. Prepare the normal growth medium and warm to 37 deg C in a waterbath.

2. Prepare 3.6% low melting point agarose (eg Seaplaque from FMC) in water. Autoclave before use,

melt in boiling water and cool to 37 deg C in waterbath.

3. Add 1 ml agarose solution (use a sterile syringe) to 9 ml warm media, mix and return to waterbath.

4. Add cells (depending on cloning efficency say 30 - 100 cells per ml). You will need to find the optimal

number that gives well isolated colonies.

5. Plate 1ml cell mix/35mm petri dish.

6. Place dishes at 4 oC for 3 - 5 minutes (on a levels surface) until agarose has set.

7. Incubate 5 - 7 days.

Methocellulose (x2)

1) Sterilize 500mL of water in a 2L conical flask containing a large magnetic stir bar.

2) Sterilize 24g of 4000 centipoises methylcellulose (#M0512, Sigma) in a 500 ml beaker.

3) Carefully remove the flask containing the water from the autoclave while still hot. Place flask on a

heated stir plate inside a laminar flow hood. Bring the water to a boil and slowly (over 15 - 30 minutes)

add methylcellulose powder with stirring. Exercise caution as addition of the methylcellulose will cause

the boiling water to foam.

4) Allow the methylcellulose solution to cool to approx 37 0C, and add 2X concentrated tissue culture

medium. Stir at 4C overnight.

Aliquot the 2X methylcellulose by pouring approximately 40mL into 50mL polypropylene tubes. Freeze

at -20C.

For use, dilute x2 methylcellulose with FCS/media supplements/x1 basal media. Vortex. Add cells and

mix by pumping up-and-down in a syringe. Allow to stand to remove bubbles. Dispense 1ml mix into 35

mm dishes.

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