Dauer purification by Sucrose Floatation and Ficoll precipitation

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Once you have a good culture with lot of dauers, you might want to separate the dauers from the non-dauers and other debris that is washed-off the worm plates. e.g. microarray studies comparing dauer vs non-dauers require as clean/pure dauer cultures as possible.

Contents

Protocols

SDS wash: To kill non-dauers

The method makes use of the fact that when ingested, SDS is toxic to the worms, but since dauers are non-feeding, they survive this treatment.

  • Reagents: 1%SDS, dH2O
  • Method:
  1. Wash off worms from plates. Resuspend worm-pellet in 50 ml 1%SDS. Incubate for 15 minutes with rocking/mixing. Longer incubation kills the dauers as well!
  2. Pellet worms to about 5ml by 3x wash/centrifuge at 1650g for 5 minutes.
  3. After the final wash, aspirate water and transfer the worm pellet to a 15ml conical tube. Proceed to sucrose flotation as below.

Sucrose Flotation: To separate live worms from debris and dead worms

  • Reagents: 60% Sucrose, dH20
  • Method:
  1. Re-suspend pellet to a final volume of about 7ml in a 15ml tube.
  2. Add 7 ml of 60% Sucrose till 14ml mark. Invert and mix well. Spin at 50g (630rpm / 660rpm) for 1min, and then immediately at 1150g (3000 rpm / 3180rpm) for 3 min.
  3. Debris settles at bottom and live worms float in the top layer/interface, usually as a yellow band. Immediately use a broad Pasteur pipette to transfer the worms in the top layer to a new 50ml conical tube. Top-up to 50ml with water.
  4. 3x wash/centrifuge at 1650g (3600 rpm / 3810rpm) for 5 min to final pellet <5ml. This pellet is almost pure dauers. For a better purification, proceed to Ficoll precipitation.

Ficoll precipitation: To further separate carcasses from live dauers

  • Reagents: 15% Ficoll in 0.1 M NaCl, dH2O
  • Method:
  1. Use Ficoll:Worms as 2:1. E.g. Take 5 ml of Ficoll in a 15ml tube. Gently layer 2.5ml dauer pellet on the top. Do not disturb. (May leave undisturbed for 10minutes to let dauers sink.)
  2. Spin at 300g (1540 rpm / 1630rpm) for 10 minutes. All live dauers pellet down. Aspirate debris from the top.
  3. 3x wash with water at ~1500g.
  4. The whole procedure is stressful to worms, so before doing any gene expression studies, let the worms recover overnight in 200-300ml of 0.1M NaCl in a conical flask, with vigorous shaking.

Dauer RNA extraction / Seeding on OP50 plates

  1. Spin down dauers at 1650g (3600 rpm / 3810rpm) for 5 min. Use dauer pellet for RNA extraction. OR : For Dauer-Exit experiments, seed 25-50ul of worm pellet on 10cm OP50 plates. Note the time of seeding.
  2. After 12 hrs of feeding dauers, wash off worms from plates and pellet by wash/centrifuge. Do a sucrose flotation if necessary.

Links

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