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Pressure-free Lineaging

Lineaging of C. elegans Embryos

by Pressure-free Mounting


Materials:

Glass Slides

18mm x 18mm Coverslips

Petroleum Jelly

#15 Disposable Scalpel

Embryonic Blastomere Medium

Procedure:

  1. Prepare coverslips with petroleum jelly spacer
    • apply thin amount of petroleum jelly to the top surface of coverslip along all four edges
  2. Dissect embryos from gravid hermaphrodites
    • pick 3-5 gravid hermaphrodites to a NG plate without E. coli
    • place coverslip ('jelly side' up) on top of glass slide
    • place 8 ul of medium on top of coverslip
    • using 32 gauge platinum pick, transfer the hermaphrodites from the unseeded plate to the drop of medium
    • cut hermaphrodites with scapel to release embryos
    • cut on both sides of the vulva to release earlier embryos and push any remaining ones out of nematode carcass by successive pressure moving in from the tip
  3. Prepare slide
    • gently place second slide on top of coverslip, allowing the medium to spread
    • apply slight pressure to edges to 'seal' the coverslip to the slide with the petroleum jelly
  4. View embryos by DIC microscopy

 

Hints & Miscellaneous

 

compiled by Chad Rappleye

Aroian Lab Protocols