- Prepare at least 5-10 small NGM plates of worms, ideally one day past starvation to enhance for L1s. If a strain is heterozygous or contains an extrachromosomal array, begin with at least 10 adults on each plate. Note: Ideally worms should be free of mold or contaminating bacteria.
- Wash worms off plates using 4-5ml S Basal into a 15 mL conical tube (want a final volume of 3mL worms in S Basal; may centrifuge to concentrate down to this volume).
- Place tube of worms on ice for a minimum of 15 minutes.
- Melt freezing agar in microwave, being careful that it is completely melted but doesn’t boil over. Place freezing solution + agar in 50°C incubator for 15 minutes
- Add your strain to the freezing logbook and label tubes with strain name and date, writing legibly with black VWR permanent marker.
- Use a 5ml disposable pipette. Pipette 3mL of molten freezing agar into tube of worms and immediately mix gently by pipetting up and down several times.
- Divide worm solution into 3 pre-labeled freezer vials.
- Freeze slowly in styrofoam block in -80°C freezer.
- Test thaw strain after about one month by scraping a small amount of frozen worm solution onto plate, using a flamed spatula that has been cooled by touching to clean ice. Keep tubes cold while doing this! Strain is considered successfully frozen if fertile worms of the correct genotype crawl out of thaw.
- Move to permanent location in liquid nitrogen and -80 after successful test thaw, and record locations in paper and digital database.
[[S Basal recipe]] - you can find S-basal on the back bench.
[[Freezing Solution recipe]] - you can find freezing solution on the back bench.
add 0.4 g bacteriological agar
add 300 ul 1M MgSO4
The final mix is 1:1 freezing solution + agar : worms in S Basal
OR JUST ASK FOR SOME READY-TO-USE FREEZING AGAR