Molecular Biology Calculator

PCR master mix

Per-reaction and batch volumes, custom or kit example preset.

What this calculator does

Calculate per-reaction and batch volumes for a PCR master mix with excess, primers and template, from custom components or a versioned kit preset.

Calculation model

Linear component scaling with excess (F04) + versioned kit preset (F16) + preparation engine

Formulas

  • F04 · Reaction and master-mix preparation: V_i = C_final,i · V_reaction / C_stock,i; V_water = V_reaction − Σ V_i; V_batch,i = N · (1 + e) · V_i
  • F15 · PCR primer stock volume: V_primer = C_primer,final · V_reaction / C_primer,stock
  • F16 · PCR master mix with template: Component scaling and water by F04; Kit values only from named, versioned presets
  • P01 · Direct working dilution: C_source · V_source = C_working · V_prepared; DF = C_source / C_working = V_prepared / V_source; V_diluent = V_prepared − V_source
  • P03 · Target transfer from selected source: V_transfer = target_amount / C_source(planned); target_amount = C_stock · V_theoretical(stock) (locked)
  • P04 · Prepared concentration after setpoint rounding: C_working,planned = C_source · V_stock,planned / (V_stock,planned + V_diluent,planned); achieved DF = (V_stock + V_diluent) / V_stock (not the requested DF if rounded)
  • P05 · Planned delivery and deviation: amount_planned = C_working,planned · V_setpoint; deviation % = 100 · (amount_planned / amount_target − 1); planned ratio = (m_i,planned / L_i) / (m_v,planned / L_v)
  • P06 · Reaction balance: V_water = V_reaction − Σ V_non-water (negative ⇒ infeasible, never clamped); DNA-only: remaining = capacity − Σ V_DNA
  • P07 · Working-solution demand and batch: demand = n_transfers · V_setpoint · (1 + excess) + dead volume; batch_i = N · (1 + e) · V_i (per-well composition unchanged)
  • P08 · Instrument setpoint quantization: setpoint_nL = round(V · 1000 / increment_nL) · increment_nL (nearest | down | up); settable ⇔ range_min ≤ setpoint ≤ range_max; increment ≠ accuracy
  • P09 · Nominal practical batch and remainder: V_batch,nom = Σ setting_j · aspirations_j (integer nL); ΔV = V_batch,nom − V_batch,theoretical; V_remainder,nom = V_batch,nom − N · V_dispense,nom (< 0 ⇒ blocked; no dead-volume model)
  • P10 · Nominal final concentration from planned settings: in mix: C_final,nom = C_source · (V_i,batch,nom / V_batch,nom) · V_dispense,nom / V_reaction,nom; per well: C_final,nom = C_source · V_i,nom / V_reaction,nom; V_reaction,nom = V_dispense,nom + Σ V_per-well,nom (nominal settings, not measured)

Assumptions

  • Volumes are additive and solutions homogeneous.
  • Excess changes the prepared batch only, not per-well concentrations.
  • Components marked “per well” (e.g. template) are added separately and not multiplied by the excess.

Verification status

  • The NEB Q5 2X Master Mix preset is an example for that product only and does not imply suitability for other polymerases.
  • Preset values were not re-checked against the live NEB page (HTTP 403 on 2026-10-29).
  • Dead volume of pipettes, tubes or dispensers is not modelled beyond the excess percentage.
  • tests-passed means automated algorithm tests (unit, property, integration and browser end-to-end) pass. It is not an independent scientific review and not laboratory validation.

References

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