TITLE: DNA Barcoding Protocol for Arthropods
AUTHORS: Valerie Warhol

[DESCRIPTION]
This is a basic protocol for doing extraction and amplification of DNA barcodes (COI) for arthropods (typically small insects or spiders).

[STEPS]
SECTION: Prepare sample and equipment
1. Make sure all instruments, such as forceps and pestle, are clean and sterile.

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 (typically 1 leg). Return specimen to freezer.
SECTION: Prepare sample and equipment
4. Let sample dry for 5–10 minutes to remove ethanol.
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 silica resin 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 wash buffer 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 wash buffer 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.
SECTION: Amplify COI (PCR)
29. For each DNA sample, label a PCR microtube with sample ID.
SECTION: Amplify COI (PCR)
30. Turn on PCR thermal cycler and connect to computer.
SECTION: Amplify COI (PCR)
31. Remove molecular grade water from refrigerator. Remove template DNA, PCR master mix, and primers from freezer. Let thaw for 10 min.

 


SECTION: Amplify COI (PCR)
32. Add 32 µL molecular grade water to each microtube.

SECTION: Amplify COI (PCR)
33. Mix forward primer by flicking. Add 1.5 µL forward primer to each microtube.

SECTION: Amplify COI (PCR)
34. Mix reverse primer by flicking. Add 1.5 µL reverse primer to each microtube.

SECTION: Amplify COI (PCR)
35. Mix template DNA by flicking. Add 5 µL template DNA to each microtube.

SECTION: Amplify COI (PCR)
36. Mix   by inverting. Add 10 µL PCR master mix to each microtube.

SECTION: Amplify COI (PCR)
37. Put microtubes in PCR thermal cycler and start touchdown PCR.
Denature: 95° C 30 sec; Anneal: 60° C 30 sec; Extend: 72° C 45 sec.
Denature: 95° C 30 sec; Anneal: 59° C 30 sec; Extend: 72° C 45 sec.
Denature: 95° C 30 sec; Anneal: 58° C 30 sec; Extend: 72° C 45 sec.
Denature: 95° C 30 sec; Anneal: 57° C 30 sec; Extend: 72° C 45 sec.
Denature: 95° C 30 sec; Anneal: 56° C 30 sec; Extend: 72° C 45 sec.
Denature: 95° C 30 sec; Anneal: 54° C 30 sec; Extend: 72° C 45 sec.
Denature: 95° C 30 sec; Anneal: 53° C 30 sec; Extend: 72° C 45 sec.
Denature: 95° C 30 sec; Anneal: 52° C 30 sec; Extend: 72° C 45 sec. Repeat this step for 28 cycles.
Final extension: 72° C 5 min.
(An initial long denature step is not needed for this protocol.)

SECTION: Amplify COI (PCR)
38. Remove microtubes from PCR thermal cycler. Verify PCR using gel electrophoresis.
SECTION: Amplify COI (PCR)
39. Store PCR products in freezer (-20 °C).