Before you start
- Collect ~2 mm tail tip or one ear punch per mouse; clean tools between animals to avoid cross-contamination.
Reagents and materials
Totals are for 24 biopsies (+10% extra). Per-biopsy amounts are in the middle column.
| Reagent | Per biopsy | Total |
|---|---|---|
| Alkaline lysis reagent (25 mM NaOH, 0.2 mM EDTA) | 75 µL | 75 µL |
| Neutralisation reagent (40 mM Tris-HCl, pH 5.0) | 75 µL | 75 µL |
Buffers and recipes
Alkaline lysis reagent per 100 mL
| NaOH (25 mM) | 100 mg (or 2.5 mL of 1 M) |
| 0.5 M EDTA, pH 8.0 (0.2 mM) | 40 µL |
| Ultrapure water | to 100 mL |
pH about 12. Store at room temperature.
Neutralisation reagent per 100 mL
| Tris-HCl (40 mM) | 0.63 g |
| Ultrapure water | to 100 mL |
Adjust to pH 5.0.
Equipment
- PCR strip tubes or 96-well plate
- Thermocycler or heat block (95 °C)
- Centrifuge
Procedure
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Add lysis reagent
Place each biopsy in a tube with 75 µL alkaline lysis reagent; make sure the tissue is submerged.
Heat
Incubate at 95 °C for 30–60 min (lid heated).
Cool
Cool to 4 °C or on ice.
Neutralise
Add 75 µL neutralisation reagent and mix.
Spin
Centrifuge briefly to pellet debris.
Use in PCR
Use 1–2 µL of the supernatant per 20 µL PCR. Store at 4 °C for weeks or at −20 °C long term.
Critical points
- Too much template inhibits PCR; try 1 µL or dilute 1:5 if bands are weak.
- Include a no-tissue control through extraction.
Troubleshooting
| Problem | What to check or change |
|---|---|
| No PCR product | Reduce template amount, check neutralisation, re-extract with a smaller biopsy. |
| Contamination in no-template control | Clean tools with bleach/ethanol between animals; use filter tips. |
Safety
NaOH is corrosive. Biopsies under your approved animal protocol.
Further reading
- Truett GE et al. Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). BioTechniques (2000).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.