C. elegans gDNA extraction.

Materials

• DNeasy Blood & Tissue Kit (Qiagen #69504)
• M9
• Ice
• Heat block set to 56°C
• Vortexer
• RNase A
• 100% ethanol

Protocol

1. 3–4 days before you plan to extract gDNA, chunk to three 10 cm plates per strain or line. Monitor plates daily and extract gDNA when plates are full of adults and nearly starved.
2. When plates are ready, wash worms off the plates and pool by strain or line into 15 mL of M9. Let settle on ice for 1 hour.
3. Aspirate off the M9 and wash with 5 mL of fresh M9.
   • Let settle on ice for ~15 minutes.
   • Aspirate the M9 and transfer worms to a 1.5 mL tube.
4. Add 1 mL M9.
   • Spin on a minicentrifuge, or let settle on ice.
   • Aspirate the M9.
5. Optional: at this point the worms can be stored at -80°C.
6. Add 180 μL of Buffer ATL and 20 μL of Proteinase K, both provided with the kit.
   • Vortex and incubate at 56°C. Check the amount of lysis after 1 hour — if you still see worms, continue the incubation; if you see embryos or nothing, proceed.
7. Add 4 μL of RNase A (100 mg/mL) and incubate at room temperature for two minutes.
8. Add 200 μL Buffer AL. Vortex and incubate at 56°C for 10 minutes.
9. Add 200 μL EtOH and vortex to mix. Transfer the contents to a labeled spin column in a collection tube.
10. Spin at 10,000 rpm for one minute. If all the contents did not go through the column, repeat the spin.