Methods
Protocol to avoid cross-contamination when disarticulating museum specimens for DNA extraction
By Arbor Ahlgren, ML Chamorro, Kojun Kanda
Ideally, to avoid cross-contamination when disarticulating museum specimens, you would work under a laminar flow hood that has never been used to process post-PCR products. The instructions provided below are for those who do not have access to such a facility. Also alternatives for sterilizing equipment: flame sterilization or UV sterilization.
Materials
1. Container for 10% bleach solution
2. Weigh boats
3. Commercial bleach
4. Gloves
5. Hype-Wipes™ or similar disinfecting towel with bleach
6. 100% ethyl alcohol in spray bottle
7. Kimwipes™ or similar delicate task wipes
8. Squirt bottle with ethanol
9. Three large tubes
10. Tall vial stand
11. Pinned specimens
12. Two sets of forceps
13. Stereoscope
14. Waterproof pen
15. Tube rack
16. Eppendorf tubes
17. Lab coat
All work surfaces and materials used must be decontaminated
Add bleach to container to make a 10% bleach solution
2. Add tap water
3. Add weigh boats to the 10% bleach solution
4. Leave weigh boats in solution for at least 5 minutes
5. Rinse weigh boats with tap water at least three times
6. Separate each weigh boat to ensure proper removal of bleach solution
7. Allow water to flow in the sink afterwards to flush the bleach from the system
8. Allow weigh boats to air dry
9. Clean all surfaces, including tables, stereoscope, and forceps
Disarticulation of specimens
10. Forceps should be cleaned and dried between disarticulation of each specimen using: a) 10% bleach solution, b) ethanol, c) distilled water
Bleach
Ethanol
Bleach
Distilled Water
Bleach
11. Select specimen
12. Remove labels with clean forceps; place in weigh boat
13. Minimally soak specimen in ethanol to remove it from card (if mounted on a card)
14. Soak up excess ethanol with a Kim wipe
15. Gently disarticulate abdomen from specimen to facilitate the buffer reaching the internal tissues (in this case, specimen is in ventral view)
16. Place all insect parts (head, thorax, and abdomen) into Eppendorf tube
17. Label Eppendorf tube with unique code.
These tubes with specimens will go through the DNA extraction process
The tubes with disarticulated specimens will be temporarily stored in a storage box for Eppendorf tubes until the DNA extraction process can take place
Re-mount all labels on the pin
Note upper left corner: all labels are on pin
18. After extraction reassociate labels and extracted specimens.