C. elegans gDNA Extraction
- Qiagen DNEasy Kit
- Heat block set to 56°C
- RNase A
- 100% ethanol
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.
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.
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.
Add 1 mL M9.
a. Spin on minicentrifuge or let settle on ice.
b. Aspirate M9.
Optional: At this point, the worms can be stored at -80°C.
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.
Add 4 μl of RNase A (100 mg/ml).
a. Incubate at room temperature for two minutes.
Add 200 μl buffer AL.
a. Incubate at 56°C with vortexing for 10 minutes.
Add 200 μl EtOH and vortex to mix. Transfer contents to a labeled spin column in a collection tube.
Spin at 10,000 rpm for one minute. Note: if all the contents did not go through the column, repeat the spin.
Remove spin column and transfer to a new collection tube. Add 500 μl Buffer AW1.
a. Spin at 10,000 rpm for one minute.
Remove spin column and transfer to a new collection tube. Add 500 μl Buffer AW2.
a. Spin at maximum speed for three minutes.
Remove spin column and transfer to a clean, labeled 1.5 ml microcentrifuge tube.
Add 200 μl Buffer AE.
a. Incubate at room temperature for one minute.
b. Spin at 10,000 rpm for one minute.
Repeat steps 13-14 and combine eluates (Buffer AE).
Measure the concentration with Qubit dsDNA BR.