C. elegans gDNA Extraction

Materials

  • Qiagen DNEasy Kit
  • M9
  • Ice
  • Heat block set to 56°C
  • Vortexer
  • RNase A
  • 100% ethanol

Protocol

  1. 3-4 days before you plan to extract RNA, chunk to three 10 cm plates per strain/line.

    a. Monitor plates daily; extract RNA when plates are full of adults and nearly starved.

  2. When plates are ready, wash worms off of plates and pool by strain/line into 15 mL of M9.

    a. Let settle on ice for 1 hour.

  3. Aspirate off M9 and wash with 5 mL of fresh M9.

    a. Let settle on ice for ~15 minutes.

    b. Aspirate M9 and transfer worms to a 1.5 mL tube.

  4. Add 1 mL M9.

    a. Spin on minicentrifuge or let settle on ice.

    b. Aspirate 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 (provided with the kit).

    a. Incubate at 56°C with vortexing. Check the amount of lysis after 1 hour. If you still see worms, continue the incubation. If you see embryos or nothing, proceed to the next step.

  7. Add 4 μl of RNase A (100 mg/ml).

    a. Incubate at room temperature for two minutes.

  8. Add 200 μl buffer AL.

    a. Incubate at 56°C with vortexing for 10 minutes.

  9. Add 200 μl EtOH and vortex to mix. Transfer contents to a labeled spin column in a collection tube.

  10. Spin at 10,000 rpm for one minute. Note: if all the contents did not go through the column, repeat the spin.

  11. Remove spin column and transfer to a new collection tube. Add 500 μl Buffer AW1.

    a. Spin at 10,000 rpm for one minute.

  12. Remove spin column and transfer to a new collection tube. Add 500 μl Buffer AW2.

    a. Spin at maximum speed for three minutes.

  13. Remove spin column and transfer to a clean, labeled 1.5 ml microcentrifuge tube.

  14. Add 200 μl Buffer AE.

    a. Incubate at room temperature for one minute.

    b. Spin at 10,000 rpm for one minute.

  15. Repeat steps 13-14 and combine eluates (Buffer AE).

  16. Measure the concentration with Qubit dsDNA BR.