bd1. Before you start
- Ethics: animal work must be approved by your institutional animal ethics committee, and euthanasia must use an approved method.
- Sterile tools: scissors, forceps, dissecting board, tips and tubes, cleaned with 70% ethanol or autoclaved. Dissect in a clean area or laminar-flow hood.
- Tubes: DNA-free 1.5 or 2 mL tubes, labelled with animal ID, date and sample type.
2. Dissection and collection
- After euthanasia, place the animal on its back on a clean surface.
- Make a midline incision from the lower abdomen to the ribcage and expose the gut without rupturing organs.
- Find the caecum (the pouch where the small and large intestines meet) and remove it with sterile scissors.
- Open it lengthwise and collect 100–300 mg of content with a sterile tip or spatula into the labelled tube.
3. Preservation
- DNA extracted within 2–4 hours: keep at 4 °C.
- Otherwise: freeze at −80 °C immediately.
- If a freezer is not available, use a preservative such as DNA/RNA Shield or RNAlater.
4. Avoiding common problems
| Problem | Solution |
|---|---|
| Contamination from tools, gloves or surfaces | Separate or cleaned tools for each animal (bleach or ethanol); change gloves often |
| DNA degradation by gut enzymes | Work quickly and on ice; freeze at −80 °C as soon as possible |
| Low DNA yield | Use stool/gut extraction kits with bead-beating (for example Qiagen, Zymo) |
| Cross-contamination between samples | Single-use consumables; one animal at a time; wipe surfaces |
| PCR inhibitors (bile salts, polysaccharides) | Kits with inhibitor removal, or an extra clean-up |
Include controls: an extraction blank and, where possible, a sample from a reference animal or environment.