This procedure allows one to obtain a sample of gDNA from an adult zebrafish. This gDNA can be used to genotype an individual by PCR. This quick and dirty preparation can be further cleaned by phenol/chloroform extractions or a commercially available kit for Southern blot analysis. However, it is not suitable for techniques that require maintenance of high molecular weight (sizes > 15-20kB) DNA.
Start =>Adult zebrafish with a healthy tail fin.
- Prepare your work area.
- Prepare a 50mL conical tube filled w/ 95% EtOH.
- Prepare a petri dish with 10% EtOH.
- Have a squirt bottle ready with H2O.
- Have a clean tweezers and scissors ready.
- Prepare a low temperature flame source (an EtOH burner-stolen from Juan- works well).
- Bring the fish to the work area in a suitable container w/ "fish" H2O.
- Clean, fill, and label an isolation chamber for each fish. This can be done one at a time, but needs to be ready to hold the fish after its tail has been clipped.
- Dip the blades of the scissors and the tweezers into the 95% EtOH and pass through the EtOH burner flame to sterilize the ends of these instruments.
- Holding a zebrafish still in a net with the tail accessible, you will want to clip approximately 1/2 to 2/3 of the end of the tail off the zebrafish shown in the image as the clip zone. Be extremely careful not to cut into the muscle of the fish shown as the tail front on the zebrafish image.
- Immediately place the clipped tail (which) usually clings to the scissors in the petri dish of 10% EtOH, and place the fish in its new isolation chamber.
- With the tweezers transfer the tail from the petri dish to labeled microcentrifuge tube.
- Use water from the spray bottle to cover the tail or to help transfer it from the tweezers to the tube.
- Place the tube into a rack at 4° C.
- Repeat steps 2 through 8 for each fish that you want to obtain gDNA from.
- Remove the H2O from each sample with a pulled Pasteur pipette.
- Add 250 µL of Tail gDNA Extraction Buffer to each sample.
- Incubate at 55° C.
- Rasp on a microcentrifuge rack or vortex every 15-20 minutes until the tail is dissolved.
- Continue incubating at 55° C for an additional hour or overnight.
- Centrifuge lysate at high speed for 5 minutes at 4° C. This step removes undissolved pigment and tail pieces from the sample.
- Transfer the supernatant to a new tube, and add 250 µL of isopropanol to precipitate the gDNA.
- Pellet gDNA by microfuging samples at high speed for 10 minutes at 4° C. Discard supernatant to alcohol waste container.
- Wash pellet with 70% EtOH. Re-spin if pellet is dislodged. Discard supernatant to alcohol waste container. Pulse samples in microfuge to collect remaining EtOH in bottom of tubes. Remove EtOH with a pipette tip.
- Allow gDNA to air dry ~5 minutes.
- Resuspend in 50 µL of TE with 20ug/mL RNaseA.
- Rasp on a microcentrifuge tube rack or vortex to resuspend pellet. Incubate at 37 °C for 30 minutes.
- Check spectrophotometer readings if desired.
- Store at -20 °C.
Tail gDNA Extraction Buffer
- 10mM Tris-Cl pH8.2
- 100mM EDTA pH8.0
- 200mM NaCL
- 0.5% SDS
- Added Fresh 200ug/mL Proteinase K