Molecular Biology Calculator

DNA ligation

Insert mass and pipetting volumes for an insert:vector molar ratio.

What this calculator does

Calculate the insert mass for a chosen insert:vector molar ratio from fragment lengths, and the pipetting volumes for the ligation reaction.

Calculation model

Molar ratio via length-scaled mass (dsDNA) + dilution-aware preparation engine (Release 2)

Formulas

  • F11 · Ligation insert mass: m_insert = r · m_vector · L_insert / L_vector; V = m / C (F10, pipetting volume)
  • F07 · dsDNA mass ↔ amount: n_pmol = 1000 · m_ng / (650 · L_bp); m_ng = n_pmol · 650 · L_bp / 1000
  • F10 · DNA mass ↔ volume: m_ng = C_ng/µL · V_µL; V_µL = m_ng / C_ng/µL
  • 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
  • P02 · Stock + diluent notation: a + b = a parts stock + b parts diluent; DF = (a + b) / a; 1 + 9 = 1:10; 10 + 190 = 1:20
  • 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

Assumptions

  • dsDNA average molar mass 650 g/mol per bp (NEB convention, [S02]); an approximation. Tools using 660 g/mol/bp give ~1.5 % different results.
  • Mass ratio scales linearly with length for equal molar amounts.
  • Measured concentrations are accurate.

Verification status

  • Calculates masses only. It does not establish the optimal ratio for your ligation or the compatibility of DNA ends.
  • Ratio choices offered are common starting points, not recommendations.
  • 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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