Tissue lysis buffer (proteinase K, genotyping) recipe
Digests tail or ear tissue with proteinase K to get DNA for genotyping PCR: 100 mM Tris pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl.
🧮 Volume calculator
| Component | Amount | Final concentration (1X) |
|---|---|---|
| 1 M Tris-HCl (pH 8.5) | 5 mL | 100 mM |
| 0.5 M EDTA (pH 8.0) | 500 µL | 5 mM |
| 10% SDS | 1 mL | 0.2% |
| 5 M NaCl | 2 mL | 200 mM |
| Proteinase K 20 mg/mL (add fresh) | 250 µL | 100 µg/mL |
| Water (distilled) | to final volume |
Steps
- Mix everything except proteinase K and store at room temperature.
- Add proteinase K just before use; use 300–500 µL per 2–5 mm piece of tissue.
- Digest overnight at 55 °C, spin, and precipitate DNA from the supernatant with isopropanol.
Good to know
- SDS inhibits PCR, so precipitate and wash — or use the SDS-free variant with 0.45% Tween-20 and 0.45% NP-40.
FAQ
Is there a faster method?
The HotSHOT alkaline-heat method gives PCR-ready DNA in about 30 minutes.
This is a widely used standard composition. Amounts depend on the hydrate form and purity of your reagents — check the formula weight (FW) on the bottle and follow your institution's safety rules and the manufacturers' instructions.