McCormick Lab Wiki

Freezing Worm Strains

1. Pick 20 young adult worms onto 10 cm plates that were seeded with 1 mL OP50. If you are working with two plates, then you should have 40 worms in total. If the worms are growing at a temperature of 15 C they will be ready to freeze within 1 week, however, if they are growing at a temperature of 20 C they will be ready in the timespan of 4 days.
2. The worms will show these signs if they are ready: There is little to no food (they are starving), there are plenty of L1s and L2s present on the plate (these are what will survive the freezing), there are eggs on the plate (it has not been long since food was gone), all the worms are of the correct phenotype, and there is no contamination on the plate
3. Using a pipette aid and a 10 mL pipette tip take 10 mL of M9 and wash the plates that are to be frozen. This can be done by placing a small amount of M9 from the tip onto the plate, swirling it (this will help loosen the worms from the agar), and tilting the plate to one side so you can pipette the liquid off the plate.
4. Each plate needs to be washed 3 times with M9 to get a good population of worms.
5. Pipette the worms and M9 into a 15 mL conical tube. There should be around 10 mL in total, however, it is fine if you are only able to pipette 8-9 mL into the conical tube.
6. Take your conical tube and centrifuge it in the tabletop centrifuge located in Summer's lab (ask nicely and they will let you use it). It should spin at 1000 rpm for 1 minute. You can do this by turning the knobs located on the centrifuge to help you configure the settings. If you need help with this, ask Christine.
7. Once the conical tube has been centrifuged, take it to the fume hood and turn on the vacuum (there should be a 50 mL conical tube covering the oil tip on it). Using the vacuum and a 1000 microliter pipette tip (with no filter), collect as much of the supernatant as you can without disturbing the “pellet” of worms.
8. Add about 10 mL of M9 into the 15 mL conical tube again and resuspend the “pellet” of worms. This is used to help clean off the bacteria that any of the worms has. Take this back to the centrifuge and set it to spin at 1000 rpm for 1 minute. Once it has been centrifuged repeat the previous step (Step 7).
9. Repeat step 8 once more without collecting the supernatant.
10. After the conical tube has been centrifuged for the third time, you will take it back to the fume hood and vacuum the supernatant again. This time, however, you will only collect enough to leave 4.5 mL of supernatant in the conical tube with the “pellet” of worms.
11. Take your conical tube back to the bench and pipette 4.5 mL of Worm freezing solution into the conical tube.
12. Screw the top on the conical tube and invert it slightly to shake the pellet into distributing the worms throughout the solution.
13. Take 9 screw top tubes and label them with the MM strain numbers located next to the -20 C Freezer. There should be on one on the side of the screw top tube, one on the top of the screw top tube, and one on the screw top tube top. Do this for all 9 tubes (All should have the same MM number, so if you are freezing one conical tube, 9 tubes should be labeled the same MM number).
14. Using a different pipette tip every time, take a P1000 pipette and pipette 1000 microliters of your worm solution into each screw top tube.
15. Locate a styrofoam container that is labeled as the “worm freezer container” within drawer 19 in the lab and place your screw top tubes in there. Cover the bottom half of the styrofoam container with the top half of the styrofoam container and rubber band it shut (2 rubber bands lengthwise, 2 rubber bands widthwise).
16. Store your worms in the -80 C freezer (bottom space) for one day, and the next day take them out and place in the worm strain box located in the top half of the -80 C freezer.
17. Once the worms are in the freezer, locate the MM-Worm-Strains Spreadsheet found on the dropbox (hint: it should be there as soon as you open the dropbox folder) and place the correct information on the spreadsheet that corresponds to the worms that you just did (state the MM number, what box it will go into, what strain they are, etc.)