C. elegans total genomic DNA preparation¶
Contributed by Ian Chin-Sang, Queens University, ON, Canada
- elegans total genomic DNA preparation
Avoid multiple freeze thaws of ProteinaseK! Either store in 50% glycerol or aliquot in small volumes and throw out after 2-5 thaws.
Worm lysis buffer: 0.1M Tris-Cl pH 8.5, 0.1M NaCl, 50 mM EDTA pH 8.0, 1% SDS. 95% EtoH and 70% EtOH (ice cold) 3M NaoAc Phenol:Chloroform (TE saturated) 20mg/ml Proteinase K (in 50mM Tris pH=8, 5mM CaCl2 and 50% glycerol (optional))
- Add 4.5 ml of worm lysis buffer to a frozen 500 ul aliquot of worms (about 5 frozen worm pellets) in a 15 ml conical tube.
- Add 200 ul of 20 mg/ml Protease K to worms and vortex. Add 100ul of 10mg/ml RNaseA (boiled to inactivate DNAses)
- Incubate at 65C for 60 minutes. Vortex 4-5 times during the incubation. The solution should clear as the worms disintegrate.
- Add 5ml of TE saturated Phenol:Chlroform (take the bottom layer) and vortex hard for 1 minute. Spin at high speed on the bench top centrifuge.
*Make sure the lid is sealed properly and wear gloves. Phenol burns can be very nasty and can even kill you. *
- Extract the aqueous (top) layer and transfer to a new 15ml tube. Do not be too greedy and leave a bit behind ensuring you do not get the phenol layer.
- Add 500ul 3M NaoAC mix. Aliquot into 300ul volumes in 1.5ml microfuge tubes.
- Add 2.5 volumes (750ul) of ice cold 95% EtOH. Invert to mix. The stringy white DNA should be obvious. Spin high speed at 4C for 5 minutes.
- Wash with 70 % EtOH. Be very careful here as this step is where most people lose the pellet.
- Dry (speed vac or air dry) and resuspend DNA in 50 ul TE for each tube and store at -80C.
Use 1-5ul in a PCR.
This method is based, with permission, on an original protocol available here.