Worm Genomic Prep

Reagents

 * Lysis Buffer
 * 200mM NaCl
 * 100mM Tris-HCl pH 8.5
 * 50mM EDTA, pH 8.0 0.5% SDS
 * -add Proteinase K to 0.2mg/mL before use (stored as 20mg/ml stock in -20°)


 * phenol/chloroform/isoamyl alcohol
 * chloroform/isoamyl alcohol
 * 3M Sodium acetate
 * 100% ethanol
 * 70% ethanol
 * TE
 * 10mM Tris-HCl, pH 8.0
 * 1mM EDTA, pH 8.0

Protocol
1. Pick N2 adult hermaphrodites onto 20 plates, 10 adults per plate, and place at 20°.

2. Wait five days until most of animals on plates are gravid adults

3. Bleach worms (see accompanying protocol) and put carcasses and eggs on 7 plates with no food, place at 20°.

4. Wait two days.

5. Rinse the worms off each plate with 2mls of M9 media and put them in 15 ml conical tubes.

6. Pellet them by spinning 2 minutes at 3000RPM in clinical centrifuge.

7. Remove supernatant and add 5 volumes of lysis buffer with Proteinase K (~100µL pellet of worms is convenient).

8. Incubate at 65°C for 1-2hrs.

9. Incubate at 95°C for 20-30min.

10. Add DNAse-free RNAse to 0.1mg/mL and incubate at 37°C 1 hour.

11. Extract twice with 1 volume phenol/chloroform/isolamyl alcohol.

12. Extract once with chloroform/isoamyl alcohol.

13. Add 0.1 volume 3M sodium acetate and >2 volumes 100% ethanol.

14. Mix and incubate at RT or in the freezer for at least 1hr.

15. Pellet DNA by centrifugation at 14000rpm for 15min.

16. Remove supernatant and wash pellet with 70% ethanol.

17. Air dry and resuspend in 200ul TE. Determine concentration and dilute if necessary.