Cryopreservation / Freezing stocks
From Wikionchus
ask Heike... & ALWAYS use Tübingen tap water!!!
Protocol
Cultivate the nematodes on one to 10 worm plates seeded with OP50. Try to obtain mostly J2 larvae as these are the ones with the best odds to survive freezing. Wash the nematodes off the plates with M9 medium using disposable Liquipettes. Collect them in 15 ml-Falcon tubes on ice. They need to be washed at least once in the same buffer by gravity sedimentation to get rid of bacteria. After washing resuspend the nematodes in 2 ml of M9, add 2 ml of sterile Freezing Solution (see below) warmed to 50°C and mix carefully. Aliquot the suspension into four cryotubes, mix and freeze immediately in styrofoam racks in a -70°C freezer. After one to two days transfer the tubes to liquid nitrogen (without the styrofoam rack!) for permanent storage. To assess the viability of the frozen stocks, thaw one aliquot and spread the contents on NGM agar plates seeded with E.coli OP50. At least 50 worms should start to move after one day.
Preparation of Freezing Solution (300 ml):
- 80 ml TÜBINGEN TAP WATER!!!
- 1.76 g NaCl
- 2.04 g KH2PO4
- 1.2 g Bacto Agar (DIFCO)
- 71.6 ml glycerol
Heat in microwave until agar is dissolved.
Fill up to 300 ml with TÜBINGEN TAP WATER.
Put 50 ml aliquots into 100 ml-glass bottles and autoclave immediately.
Cool down to 60°C and add 1.5 ml of 0.1 M Mg2SO4.
Keep at room temperature.
Before using the Freezing Solution heat it up in the microwave for 30-60 sec. Do not boil it!!! After shaking bubbles should be visible. Equilibrate the solution in a 50°C water bath.
Links
- Back to Pristionchus pacificus Protocols page
