HotSHOT alkaline lysis reagent recipe
The first reagent of the HotSHOT method, which yields genotyping-PCR DNA in about 30 min by heating tissue in alkali: 25 mM NaOH, 0.2 mM EDTA.
🧮 Volume calculator
| Component | Amount | Final concentration (1X) |
|---|---|---|
| 10 M NaOH | 250 µL | 25 mM |
| 0.5 M EDTA (pH 8.0) | 40 µL | 0.2 mM |
| Water (distilled) | to final volume |
Steps
- Mix NaOH and EDTA in water (store at room temperature).
- Add 75 µL to a 1–2 mm tissue piece and heat at 95 °C for 30 min.
- Cool, add an equal volume (75 µL) of neutralisation reagent, and use 1–2 µL of supernatant per PCR.
FAQ
Is the DNA yield enough?
It is lower and less pure than purified DNA, but plenty for short genotyping amplicons (<1 kb).
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.