C. elegans genomic DNA prep
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
(~100L 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.
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