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Freezing Worm Stocks

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1. Grow 3-5 6cm. plates of worms on NGM with OP50 until the plate is starved with lots of young larvae. The L1s are the ones that survive freezing the best and are the most likely to be fertile upon thawing.

2. Warm freezing buffer (FB) to room temperature.

3. Label five CRYOSTORAGE tubes for each stock to be frozen with the strain's ID# and the date. Capsicles fit into the tops of the cryostorage tubes. Label both the top and the side of each tube.

4. Wash worms from one plate to the next with the FB using a sterile transfer pipet to transfer worms and buffer. Use a new pipet for each worm stock and be careful not to contaminate the FB stock with worms.

5. Aliquot a 0.5ml. volume of worms into each of four labelled cryostorage tubes.

6. Put the tubes of worms into a styrofoam freezing box and put at -80C for at least 24 hours. Label the box with the date and your initials.

7. Transfer tubes to liquid nitrogen tanks and fill out the appropriate paperwork for storage records.

8. Thaw a tube to check if the worms survived the freezing process. Worms must be regrown before they can be frozen again (i.e. the same worm will not survive being frozen twice).

Freezing Buffer:

Mix NaCl (2.93 grs)

KH2PO4 (3.4 grs)

Glycerol (30% vol)

NaOH (2.8 mls).

Take to 500 ml with distilled water.

Autoclave for 20 minutes.

Allow to cool to about 60 C.

Add 3 mls MgSO4 sterilely.

Aliquot into sterile 50 ml. culture flasks.

Store at 4C.