Techniques: DNA extraction

In the past DNA extraction was accomplished using a salting out protocol adapted from Sunnuks and Hales, 1996 (MBE 12:510-524) - see below for this process. DNA is now extracted using DNAeasy Blood and Tissue Kits from Qiagen Corp. (Cat. No. 69506) incubating specimens in extraction buffer + proteinase K for 6-24 hours and eluting DNA with 50-150 µl of the supplied elution buffer.

Former protocol:

1. Place containers of 100% and 70% ethanol in the freezer.
2. Remove insect specimen from its tube using flame-sterilized tweezers. Place it into a weighboat to air dry, covered with a kimwipe. If it's a large specimen, you may want to use only legs, that wll also allow the ethanol to evaporate more rapidly.
3. Make mortar and pestles by melting the end of a 1000 µl pipet tip in a flame and then squashing it into a 1.5 ml eppendorf tube.
4. Mix proteinase K and TNES in a 1:60 proportion (5 µl of 10mg/ml proteinase for every 300 µl TNES: 50mM Tris ph 7.5, 400mM NaCl, 20 Mm EDTA, 0.5% SDS).
5. Put specimen into one of the above tubes.
6. Pour liquid nitrogen into the tube (makes the specimen easier to crush), then squash with the pestle.
7. Pour 305 µl of the TNES/proteinase K mixture down the pestle to wash as much squished insect possible into the tube.
8. Vortex briefly.
9. Incubate tube at 37 ºC overnight or 55 ºC for 3 hours. Vortex frequently (every 15 minutes) if possible.
10. Add 85 µl 5M NaCl (saturated solution) and invert tube to mix (proteins precipitate).
11. Centrifuge at 14,000 rpm for 5 minutes (put tubes with hinge facing out).
12. Move the liquid to a new labelled 1.5 ml tube.
13. Add one volume of cold 100% ethanol to the new tube with supernatant. Mix by inverting tube.
14. Spin the tube at 14,000 rpm for 5 minutes, again with hinges outward.
15. Use a pipet to dispose of the supernatant, being careful to avoid the DNA deposited on the bottom and hinge side of the tube.
16. Add 500 µl 70% ethanol.
17. Spin the tube at 14,000 rpm for 5 minutes, again with hinges outward.
18. Remove the ethanol as above.
19. Spin for 2 seconds more, remove the last ethanol.
20. Let tubes air-dry (lids open, covered with kimwipe).
21. Add 50 µl of TE, keep at room temperature for 1 hour to resuspend the DNA.
22. Place in freezer.