Nematode DNA extraction methods from Barbara Wimmer

I have tried several methods for extracting from long-term ethanol preserved nematodes and none of the more common ones worked to get PCRable DNA; the GeneReleaser Protocol seems to work in most cases.

Extraction method for single nematodes with GeneReleaser

(Bioventures, Canada; can be obtained from Cambio Cat.No. BV-1157-10 (100 Assays for £20) or BV-1157-40 (400 Assays for £75)).

Pick single worm in 20 µL of PCR buffer and put at 70°C for 10 minutes

Digest with 2 µL Prot. K (10 mg/ml) for at least three hours at 56°C

Add 20 °C GR

Microwave for 6 minutes, if microwave has 750 watts; adjust accordingly, if microwave has more or less power (optimum: 4500 watt-minutes);

NOTE: Make sure that your tubes are microwaveable! Put 40 µL of water in a tube and microwave at the same settings you are going to use with your samples

Centrifuge and transfer supernatant to a new tube. (Although unlike Chelex, GR raisins do not inhibit PCR)

Pick single worm in 20 µL of PCR buffer and put at 70°C for 10 minutes

Add 20 °C GR

Microwave for 6 minutes, if microwave has 750 watts; adjust accordingly, if microwave has more or less power (optimum: 4500 watt-minutes)

Centrifuge and transfer supernatant to a new tube

Works as well, but success rate for PCRs might be lower