Why the lysis step is the bottleneck
Garbage in, garbage out
Mouse genotyping reliability is set by the lysis step, not the PCR. A clean, fully digested lysate gives a single bright band; a partially digested or over-diluted lysate gives a smear, missing band, or false homozygote call. Most "this PCR is broken" tickets in a busy colony trace back to the lysis volume being off, the proteinase K being old, or the tissue being too small. Standardizing the per-sample volumes is the single biggest reliability win.
Why three recipes
No single buffer is best for every situation. DirectPCR is the fastest (one tube, no neutralization, no precipitation), NaOH/Tris is the cheapest (no proteinase K), and proteinase K + standard lysis is the most flexible (handles bone, cartilage, toes, and gives DNA you can store and re-amplify). Most labs converge on one workhorse and keep the other two as fallbacks.
Buffer is fungible, proteinase K is not
Once a proteinase K stock is thawed it loses ~10% activity per freeze-thaw cycle. Aliquot fresh stock into single-use 50 µL tubes and pull a fresh aliquot for every master mix. The per-sample volumes the calculator outputs assume fresh, 100%-active enzyme.
Where the three recipes come from
1. DirectPCR (Viagen Biotech)
A buffered surfactant mix sold by Viagen as DirectPCR Lysis Reagent (Tail or Cell formulation). The published per-sample protocol is 200 µL of buffer + 0.4 µL of proteinase K from a 1 mg/mL working stock per 2-5 mm tail snip, incubated at 55 °C overnight followed by 85 °C × 45 min to inactivate the enzyme. The 1-2 µL of crude lysate that goes directly into the PCR is the convenience win — no precipitation, no neutralization.
2. NaOH / Tris-HCl ("hot shot")
Truett, Heeger, Mynatt, Truett, Walker & Warman published this in BioTechniques 29:52-54 (2000) under the name "HotSHOT". The recipe is 75 µL of 25 mM NaOH + 0.2 mM EDTA per ear punch, 95 °C × 30-60 min, then 75 µL of 40 mM Tris-HCl pH 5.5 to neutralize. No proteinase K, no SDS, no precipitation. It is the cheapest published mouse genotyping lysis and remains the default for high-throughput core facilities.
3. Proteinase K + standard lysis buffer
A descendant of the Laird et al. (1991) tail-DNA prep — 100 mM Tris-HCl pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, with proteinase K added fresh to 0.2 mg/mL final concentration. 200 µL of buffer + 5 µL of 10 mg/mL proteinase K per sample, incubated at 55 °C overnight, then 95 °C × 10 min to inactivate. With an optional NaCl/isopropanol precipitation step you get DNA clean enough to re-amplify, sequence, and store at -20 °C indefinitely.
