Preparation of C. elegans Genomic DNA
Protocol by Andy Fires Lab
Modified my Min-Ho Lee and Sudhir Nayak
This protocol is based on the original Fire lab protocol for isolating genomic DNA from C. elegans, which is used as carrier for complex transgene arrays generated through microinjection or for Next Generation DNA sequencing. It has been modified to allow for large scale preparation of genomic DNA from C. elegans (or related nematodes). If you want a small scale prep, take a look at the original protocol or scale this one down.
1) Worm lysis buffer; 0.1M Tris-Cl pH 8.5, 0.1M NaCl, 50 mM EDTA pH 8.0, 1% SDS. Store at RT. There could be some precipitation after long term storage. If there is any precipitate then put it at 37C before use.
2) CTAB/NaCl solution; 10 % CTAB in 0.7M NaCl
Dissolve 4.1 g NaCl in 80 ml distilled water and slowly add 10 g CTAB (Sigma M-7635) while heating and stirring. If necessary, heat to 65C to dissolve. Adjust final volume to 100 ml.
3) Protease K; 20 mg/ml in TE pH 8.0. Store at -20C in single use aliquots. DO NOT FREEZE-THAW.
1) Seed 4 x 10 cm plates with 15 young adult hermaphrodites and grow 1 week to have a lot of starved L1.
2) Harvest starved L1 from plates and do 200 ml size liquid culture for 3-4 days at 20C until the majority of worms reach the adult stage.
3) Harvest and do 30% sucrose float twice to remove E. coli and other contaminants.
4) Aliquot worms in 500ul volumes in 1.5 ml Epp. tubes and freeze at -80C.
NOTE: You can also grow worms on 10cm plates (or egg powder plates) instead of using liquid culture.
1) Add 4.5 ml of worm lysis buffer to a frozen 500 ul aliquot of worms. (transfer to 15 ml Falcon tube)
2) Add 200 ul of 20 mg/ml Protease K to worms and vortex.
3) Incubate at 62C for 60 minutes. Vortex 4-5 times during the incubation. The solution should clear as the worms disintegrate.
4) Add 800 ul of 5M NaCl. Mix thoroughly by inversion (important)
5) Add 800 ul CTAB solution. Incubate 10 minutes at 37C.
6) Add 7 ml chloroform, mix and spin. Recover aqueous phase.
7) Add 7 ml phenol/chloroform/isoamyl alcohol (saturated with TE pH 8.0), mix and spin. Recover aqueous phase.
8) Add 0.6 volume of -20C isopropanol. Invert to mix. The stringy white DNA should be obvious. Spin at 4C for 5 minutes.
9) Wash twice with 70 % EtOH
10) Dry and resuspend DNA in 340 ul TE.
11) Add 10 ul of RNase A (10 mg/ml heat-treated to kill DNase) and incubate 2 hours at 42C.
12) Add 20 ul of 20 % SDS, 10 ul of 0.5 M EDTA pH 8.0, 20 ul of Protease K and incubate 65C for 2 hours.
13) Add 40 ul of 10M Ammonium Acetate.
14) Extract twice with phenol/chloroform/isoamyl alcohol (saturated with TE pH 8.0), once with chloroform. Add 1 ml of EtOH, mix and spin at 4C for 10 minutes.
15) Wash twice with 70% EtOH.
10) Dry and resuspend DNA in 200 – 500 ul TE. (usually get 2 to 5 mg/ml genomic DNA. Should run agarose gel to make sure the size is big)