TITLE: Inexpensive DNA Extraction Protocol
AUTHORS: Valerie Warhol

[DESCRIPTION]
This is a protocol for doing extraction of DNA using inexpensive reagents rather than enzymes or kits. It can be used for plant, animal, or fungus samples.

[STEPS]
SECTION: Prepare sample and equipment
1. Make sure all instruments, such as forceps and pestle, are clean and sterile. Make sure   has been diluted 1:1 in 95% ethanol.

SECTION: Lyse cells
6. Put sample in tube. Grind sample with pestle until broken up into tiny pieces.
SECTION: Prepare sample and equipment
2. Prepare water bath at 65 °C.
SECTION: Prepare sample and equipment
3. Dissect sample from specimen. (This will be a piece of tissue approximately 10–20 mg. For small arthropods such as beetles or spiders, 1 or 2 legs will typically suffice.) Return specimen to freezer.
SECTION: Prepare sample and equipment
4. If sample was stored in ethanol, let sample dry for 5–10 minutes.
SECTION: Prepare sample and equipment
5. Prepare a clean 1.5 mL hinged tube by writing sample ID on it and filling with 250 µL of  

SECTION: Lyse cells
7. Incubate sample tube in 65 °C water bath for 10 min.

SECTION: Lyse cells
8. Remove tube and lower temperature of water bath to 57 °C.

SECTION: Lyse cells
9. Centrifuge tube for 1 min at maximum speed to pellet debris.

SECTION: Lyse cells
10. Remove    from refrigerator.

SECTION: Lyse cells
11. Label a clean 1.5 µL tube with sample number. Transfer 150 µL of the supernatant to the clean tube. Discard old tube containing debris.

SECTION: Bind DNA
12. Add 3 µL of    to tube. Mix well by pipetting up and down several times.

SECTION: Bind DNA
13. Close tube and incubate for 5 min in 57 °C water bath.

SECTION: Bind DNA
14. Centrifuge for 30 s at maximum speed to pellet the resin.

SECTION: Bind DNA
15. Use a pipette with a fresh tip to remove the supernatant, being careful not to disrupt the pellet.
SECTION: Wash
16. Remove molecular grade water from refrigerator and   from freezer.

SECTION: Wash
17. Add 500 µL of ice-cold    to the pellet. Mix well by pipetting up and down several times to resuspend the silica resin.

SECTION: Wash
18. Close the tube and centrifuge for 30 s at maximum speed to pellet the resin.

SECTION: Wash
19. Use a pipette with a fresh tip to remove the supernatant, being careful not to disrupt the pellet.
SECTION: Wash
20. Again, add 500 µL of ice-cold    to the pellet. Mix well by pipetting up and down to resuspend the silica resin.

SECTION: Wash
21. Close the tube and centrifuge for 30 s at maximum speed to pellet the resin.

SECTION: Wash
22. Return wash buffer to freezer.
SECTION: Wash
23. Use a pipette with a fresh tip to remove the supernatant, being careful not to disrupt the pellet. Spin the tube briefly to collect any remaining drops of supernatant, and then remove these with a pipette.
SECTION: Elute DNA
24. Add 100 µL of molecule grade water to the silica resin and mix by pipetting up and down several times.

SECTION: Elute DNA
25. Incubate the mixture at 57 °C for 5 minutes.

SECTION: Elute DNA
26. Centrifuge for 30 s at maximum speed to pellet the resin.

SECTION: Elute DNA
27. Label a clean 1.5 µL tube with sample number. Transfer 90 µL of the supernatant to the clean tube, being careful not to disturb the pellet. Discard old tube containing the resin.

SECTION: Elute DNA
28. Store sample in freezer until ready to PCR. If going directly to PCR, put sample in refrigerator.