C. elegans Total Genomic DNA Isolation


Solutions

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)) Avoid multiple freeze thaws! Either store in 50% glycerol or aliquot in small volumes and throw out after 2-5 thaws.

 

DNA preparation

1) 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.

2) Add 200 ul of 20 mg/ml Protease K to worms and vortex.

2b) Add 100ul of 10mg/ml RNaseA (boiled to inactivate DNAses)

3) Incubate at 65C for 60 minutes. Vortex 4-5 times during the incubation. The solution should clear as the worms disintegrate.

4) Add 5ml of TE saturated Phenol:Chlroform (take the bottom layer) and vortex hard for 1 minute. Make sure the lid is sealed properly and wear gloves. Phenol burns can be very nasty and can even kill you. Spin at high speed on the bench top centrifuge.

5) 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.

6) Add 500ul  3M NaoAC mix. Aliquot into 300ul volumes in 1.5ml microfuge tubes.

7) 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.

8) Wash with 70 % EtOH. Be very careful here as this step is where most people lose the pellet.

9) 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.