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PCR Master Mix Scaler

Compute per-reaction and scaled master-mix volumes for Taq PCR from stock/final concentrations. Flags sub-0.5 µL pipetting precision issues and exports a bench-ready protocol with component addition order.

Mouse Colony ManagementGenotypingClient-Side

Try it out

Load example PCR master mix scaler data to see the full workflow

Batch

Component Concentrations

Typically 10× stock → 1× final.

Stock mM

Final mM

Stock µM / Final µM

Stock µM / Final µM

Stock (U/µL)

U per reaction

Added per tube (NOT in master mix).

Master Mix Recipe

ComponentStockFinalPer rxn (µL)Master mix (µL)Flag
10× PCR buffer10 ×1 ×2.500132.0
dNTP mix10 mM0.2 mM0.50026.4
MgCl₂25 mM1.5 mM1.50079.2
Forward primer10 µM0.5 µM1.25066.0
Reverse primer10 µM0.5 µM1.25066.0
Taq polymerase5 U/µL0.04 U/µL0.20010.6< 0.5 µL
Water——16.800887.0
Template (per tube)——1.000(per tube)

Summary

Total master mix
1.27 mL
52.8 effective reactions (incl. 10% overage)
Aliquot per tube
24.0 µL
+ 1.0 µL template = 25 µL total
96-well plates
1
48 wells on last plate
Warnings
  • • Taq polymerase: per-reaction volume 0.200 µL is below the 0.5 µL pipetting precision floor. Pre-dilute the stock or scale up the reaction volume.

Bench Protocol

PCR master mix for 48 reactions × 25 µL (10% overage included).

Master mix (combine in order):
  • 887.0 µL nuclease-free water
  • 132.0 µL 10× PCR buffer (10 × stock)
  • 26.4 µL dNTP mix (10 mM stock)
  • 79.2 µL MgCl₂ (25 mM stock)
  • 66.0 µL Forward primer (10 µM stock)
  • 66.0 µL Reverse primer (10 µM stock)
  • 10.6 µL Taq polymerase (5 U/µL stock)

Aliquot 24.0 µL master mix into each tube/well.
Add 1.0 µL template DNA per tube (final volume: 25 µL).

Per-reaction breakdown:
  10× PCR buffer         2.50 µL  (1 × final)
  dNTP mix               0.50 µL  (0.2 mM final)
  MgCl₂                  1.50 µL  (1.5 mM final)
  Forward primer         1.25 µL  (0.5 µM final)
  Reverse primer         1.25 µL  (0.5 µM final)
  Taq polymerase         0.20 µL  (0.04 U/µL final)
  Water                 16.80 µL
  Template               1.00 µL  (added per tube)
                       ──────
  Total                 25.00 µL
  • Setting up genotyping PCR for a batch of tail snips or ear punches
  • Switching reaction volume (e.g., 25 µL to 10 µL for screening) and needing re-scaled component volumes
  • Onboarding a new tech who needs a printable protocol with exact volumes
  • Auditing a failing PCR — re-derive the expected volumes and compare against what was actually pipetted
  • Justifying reagent reorder quantities with exact per-reaction and per-batch usage

Don't use for

  • For qPCR / real-time PCR — those have dye-specific master mixes with different component ratios
  • For pre-mixed commercial master mixes (e.g., GoTaq Green) where you just add primers + template
  • For high-fidelity cloning PCR (Phusion, Q5) — different Mg²⁺ and extension-time requirements
  • For RT-PCR — reverse transcription has its own enzyme, buffer, and dNTP requirements

Why scale via a master mix?

The case for a shared tube

Pipetting 6 components into 48 individual tubes is 288 pipetting steps. Pipetting 6 components into one master-mix tube and aliquoting once is 54 steps — a 5× reduction in opportunities for error. Every extra pipetting step adds 1-3% CV on top of whatever concentration you are targeting, so the master-mix approach is not just faster but also more precise.

Template is the exception

Template DNA (the crude tail lysate or purified genomic DNA) varies per sample by definition. It gets added after aliquoting, one tube at a time. The calculator excludes it from the master-mix math and tells you the per-tube aliquot + per-tube template volume as a pair.

Water goes in first

Add nuclease-free water to the tube first, then buffer, then dNTPs, then MgCl₂, then primers, then enzyme last. The enzyme goes last because it is the most expensive component and the most vulnerable to denaturation if it sits in a low-salt, high-temperature environment. The bench protocol in this calculator lists components in recommended addition order.

Common PCR failures traced to the master mix

No band (no amplification)

1. dNTP concentration too low — 0.2 mM (each) is standard; dropping to 0.05 mM starves the polymerase. 2. Template volume too high — more than 10% of the reaction volume introduces too many lysate inhibitors (SDS, proteinase K, salt). 3. MgCl₂ omitted when the buffer is MgCl₂-free — uncheck the box only if your buffer truly includes it.

Multiple non-specific bands

1. MgCl₂ too high — try 1.0-1.5 mM instead of 2.5 mM. 2. Primer concentration too high — drop from 0.5 µM to 0.2-0.3 µM final. 3. Annealing temperature too low — not a master-mix issue but frequently misdiagnosed as one.

Faint band on the expected lane

1. Template too little — check tissue lysis completeness. 2. Old proteinase K in the lysis step upstream (see the Tissue Digestion Volume Calculator). 3. Pipetting precision on the Taq aliquot — if you are pipetting 0.2 µL, a 20% error means ±0.04 µL, which can halve the enzyme in some tubes. Pre-dilute to a working stock.

Frequently Asked Questions

Next Steps

ConductColony — print PCR work orders for every batch of tail snips

Auto-generates master-mix recipes, plate maps, and primer-lot tracking for every genotyping batch. Links each PCR result back to the tissue lysis step.

Open app →

Tissue Digestion Volume Calculator

Scale lysis buffer volumes for the tail snips that feed into this PCR — the upstream step in the genotyping workflow.

Browse →

Mendelian Ratio & Chi-Square Calculator

After the gel: test whether your observed genotype ratios match Mendelian expectations.

Browse →

Trio Breeding Scheme Planner

Plan the breeding scheme that produces the weanlings you are genotyping.

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