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HP-30 Age Sync

Plate Staging

Optimization Assay

  1. Using stock plates that are not more than 1 month old, chunk to a fresh labeled 6 cm plate.
  2. After 48 hours, spot bleach gravid adult animals. Look for males and bacterial contamination. If either is found, then clean the culture and pick L4s to remove males.
  3. After 12-15 hours, pick L1s from the bacterial lawn away from the bleach to a fresh 6 cm plate. Pick 20-30 L1s per genotype.
  4. After 48 hours, pick 4-5 L4s to a fresh 6 cm plate. Pick 1-2 plates per genotype.
  5. Three days later, pick 5 L4s to a fresh 6 cm plate. Pick XX plates per genotype.
  6. Four days later, you will be ready to perform a filtering assay. Before filtering, it is best to pick 5 L4s to ~5 6cm plate. These plates can be used in the next optimization assay 4 days later. Repeat this step throughout optimization process in order to save time and plates.

**** Take care to avoid using plates with any visible bacterial or fungal contamination as this contamination could get propagated through to the next steps ****

Scaling Assay

  1. Using stock plates that are not more than 1 month old, chunk to a fresh labeled 6 cm plate.
  2. After 48 hours, spot bleach gravid adult animals. Look for males and bacterial contamination. If either is found, then clean the culture and pick L4s to remove males.
  3. After 12-15 hours, pick L1s from the bacterial lawn away from the bleach to a fresh 6 cm plate. Pick 20-30 L1s per genotype.
  4. After 48 hours, pick 4-5 L4s to a fresh 6 cm plate. Pick 1-2 plates per genotype.
  5. Three days later, pick 5 L4s to a fresh 6 cm plate. Pick XX plates per genotype.
  6. After ~96 hours, the culture will have many gravid animals but not be starved. You are ready to filter.

**** Take care to avoid using plates with any visible bacterial or fungal contamination as this contamination could get propagated through to the next steps ****

Filter Cleaning

** **Clean filters right before you plan to use them ****

  1. Place filters in 10% bleach solution for 10 mins. Stir occasionally.
  2. Briefly rinse filters in 70% ETOH.
  3. Use compressed air to dry filters.
  4. Place dry filters on a piece of aluminum foil.
  5. Wrap the clean filters in aluminum foil.

Filter Stack Set up

  1. For each strain, set up a single 50 mL conical tube with a connector ring (in the closed position) attached to a 30 µm filter attached to a funnel

Filtering

  1. For each plate in your strain prep:
    1. Add ~2mL of M9 to plate. Vigorously swirl and tap plate to remove larger adults from agar. Discard M9 worm solution. Repeat 5 times. (If you are also performing 40-40-20 Age Synch you can pour M9 worm solution onto filter stack)
    2. Prepare a hockey stick using a disposable glass pipette (make a new hockey stick for each strain in your prep).
    3. To collect the embryos, add a small amount of M9 to your plate and use the hockey stick to break up the bacterial lawn.
    4. Add the M9 & embryo solution to your filter stack.
    5. To ensure all the embryos are collected, add ~2mL of M9 to the plate. Swirl and pour into filter stack. Repeat twice.
    6. Release the filter valve. You may have to gently tap the filter stack on the lab bench to get the fluid to flow.
    7. Repeat these steps for each plate in your prep.
  2. Once you have finished with all the plates in your prep, rinse the filter stack. Add ~3mL of M9. Release the filter valve and allow all of the liquid to flow through. You may have to gently tap the filter stack on the lab bench to get the fluid to flow. Repeat 3 times.
  3. After washing, use a 3mL syringe to ensure all liquid has traveled through the filter. Remove the red filter valve and attach the syringe. Pull back the plunger to aspirate the filter stack.

Collecting Embryos

**** When collecting embryos it is important to use K-medium ****

  1. To collect the embryo population set up a new stack containing: (a) a fresh 15 mL tube, (b) a glass funnel, (c) the 30 µm filter, inverted
  2. Fill a 10mL syringe with K-media and attach 1.5 gauge needle. Squirt the K-media on to the underside of the 30 µm filter, allowing embryos to leave the filter and enter the 15 mL conical through the glass funnel.
  3. To pellet the embryos, spin the 15mL conical at 1100 RPM for 1 minute.
  4. Aspirate down to 2mL of K-media

Plating Embryos

** If you are performing an optimization assay you do not have to titer embryos **

Optimization Assay

  1. If you are performing an optimization assay you do not have to titer your embryos. Instead, vortex the 15 mL conical for ~10 seconds. Pipette 400 µL of embryo solution into a 96 well plate.

Scaling Assay

  1. Determine the titer of embryos by counting the number of embryos in 3 µL. Adjust the volume of K-media to obtain the desired concentration (~0.5 - 1 embryo per µL).
  2. Vortex each 15 mL conical of embryos for at least five seconds before pouring into single channel reservoir. Either pipette immediately or use the mix function before pipetting into assay plates. Pipette 50 μl of embryo solution to get to about 25-50 animals into the number of tissue-culture treated 96-well flat-bottom plates that are needed for your assay. Use the Eppendorf Xplorer 12-channel repeat pipetter to speed up the process and decrease repetitive strain. Remember to mix each row three times before aspirating and dispensing to tissue-culture plates. Use the 12-channel pipet set to 100 μL to mix.
  3. Seal all 96-well plates with Rayon gas permeable strip (Fisher cat #14-222-043). Try to avoid wrinkles in the Rayon strip.
  4. Shake overnight at 180 rpm at 20ºC on the Excella E24R shaker in a freshly made humidity chamber.
    1. To make a humidity chamber, place damp paper towels in a clean (bleached and rinsed) plastic box, close the box, and seal all around the top lid edge with parafilm.

Feeding Strains

**** You only need to feed if you are performing a Scaling Assay ****

  1. Make food from bacterial lysate (recipe at bottom of this protocol) or frozen HB101 previously prepped (see protocol). You must feed worms around the same time you will run the plates on the sorter two days later. If you’re running many plates, start making food early in the morning.