Genomic DNA Extraction from fish fin clips

1. Proteinase K digestion

Use about a 10:1 volume:volume ratio of extraction buffer to tissue. Typically we use 200 ul of extraction buffer and 1-2 mm2 of fresh dorsal fin. To 200 ul of extraction buffer, add 4 ul of 20 mg/ml Proteinase K. Add the tissue and place in 37 C water bath. Leave for 2-3 hours until tissue has disintegrated (can shake every hour or so).

For ethanol preserved finclips, use a slightly larger piece (4-5 mm long). Remove the fin and dry in speed vac briefly. Put in 200-500 ul extraction buffer. Add 4-10 ul Proteinase K (depending on extraction buffer volume). Leave at 37C till tissue is dissolved.

2. Phenol chloroform extraction

Prepare a Phase lock gel tube (3'-5') by spinning down gel in microfuge (1 min 12000 rpm). Add extraction solution (volume V). Add an equal volume of phenol (taking phenol off the bottom of tris buffered phenol solution). Invert tube several times. Spin (all spins for extraction are 1-2 min at 12000 rpm in a room temp microfuge).

Add volume V/2 of phenol and V/2 of chloroform to the tube (this makes 1:1 phenol chloroform added in volume equal to initial extraction volume). Spin again.

Add volume V of chloroform to tube. Spin again.

ALTERNATIVE: For larger volumes, you can use the blood tubes which can handle several ml. The agar in these tubes can not take full strength phenol. Instead of phenol followed by 1:1 phenol:chloroform, do two extractions with 1:1 phenol:chloroform (skipping the straight phenol step).

3. Ethanol precipitation

Transfer aqueous layer remaining above the gel to a clean tube. Determine its volume. Add 1/10 volume of 3M NaOAc and 2 volumes of 100% ethanol. Put in freezer for at least an hour (can leave overnight if need be).

Spin for 30 min at 4C, 12000g (11000 rpm in Hermle). Pipette off ethanol. Add 200 ul of cold 70% ethanol. Spin 5 min at 4 C, 12000g. Pipette off ethanol.

Dry in speed vac for 15 min. Resuspend in about 200 ul TE (can also use TE/10 but not water).

4. DNA characterization

Quantify DNA with spectrafluorometer. Run 100-200 ng on a 1% agarose gel. DNA should be high molecular weight (single band above 12kb band of 1 kb size standard) and show no evidence of low molecular

KC 3/02