This ligation calculator converts a desired insert:vector MOLAR ratio into nanograms: ng(insert) = ratio × ng(vector) × (insert length ÷ vector length). Molar ratios are what ligase sees; nanograms are what your pipette sees — this page translates between the two worlds.

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Why Nanograms Lie and Moles Don’t

Ligation is a molecular collision business: what matters is how many insert ENDS meet how many vector ENDS — counts of molecules, i.e., moles. Equal nanograms of a 1.5 kb insert and a 5 kb vector are NOT equal molecules (the insert prep holds 3.3× more molecules per ng), which is why protocols specify molar ratios and why this translation exists. The defaults: 100 ng of a 5.0 kb vector at a 3:1 ratio needs 3 × 100 × (1.5/5.0) = 90 ng of insert — 30 fmol of vector against 91 fmol of insert (dsDNA averages 660 g/mol per base pair, the constant the fmol check in the result uses). Skipping the size correction is the classic beginner error: “100 ng + 100 ng at 3:1” actually delivers ~1:1 when the insert is a third the vector’s length — and the colonies come up empty or vector-only for reasons that feel like fate but are arithmetic.

Bench context around the number: total DNA concentration shapes the reaction’s topology — dilute ligations favor intramolecular circles (the desired plasmid) over intermolecular concatemers; typical totals run 10–200 ng in 10–20 µL. Vector self-ligation is fought with dephosphorylation (CIP/SAP) and fought again by the ratio itself: every insert end occupying a vector end is a vector that cannot simply recircularize. Controls that professionals run alongside (vector-only ligation, uncut vector transformation) diagnose which failure occurred when colonies disappoint — the ratio math assumes competent ends, so gel-verify both digests before blaming the calculator’s department.

Worked Examples (Verified)

  • Defaults: 3 × 100 × (1.5/5.0) = 90.0 ng insert; 30 vs 91 fmol.
  • Big insert, small vector: 3 kb insert into 2.7 kb vector, 50 ng vector, 3:1 → 3 × 50 × (3/2.7) = 166.7 ng — insert mass EXCEEDS vector mass at equal ratio logic; correct, and a surprise only to the ng intuition.
  • Blunt-end rescue: same defaults at 7:1 → 7 × 100 × 0.3 = 210 ng — blunt ligation’s inefficiency is paid for in insert excess.

Ligation Discipline

  • Quantify both DNAs properly (fluorometric, not just A260 on a dirty prep): the ratio is only as real as the ng figures fed into it — our DNA Concentration Calculator handles the spectrophotometric side.
  • Gel-purify digests when a stuffer fragment exists: a 0.5 kb stuffer at “insert” molarity is a competing insert with its own ratio claim.
  • Fresh ATP buffer and ligase that hasn’t freeze-thawed ten times: the ratio optimizes collisions, not dead enzyme.
  • For Gibson/HiFi and Golden Gate assemblies the same mole logic applies with their own stoichiometries (equimolar fragments for Gibson overlaps; the formula here adapts by setting ratio = 1 per fragment).
  • Two-insert ligations extend pairwise: compute each insert against the vector at its own ratio, and keep the total DNA in the sane band.

Related Tools

Frequently Asked Questions

How do you calculate insert amount for a ligation?

ng(insert) = molar ratio × ng(vector) × (insert kb ÷ vector kb). Defaults: 3 × 100 ng × (1.5/5.0) = 90 ng. This calculator returns insert ng, total DNA, and the femtomole counts of both species as a mole-ratio audit.

What molar ratio should I use?

3:1 insert:vector is the standard starting point for cohesive-end cloning. Blunt-end ligations are less efficient — 5:1 to 7:1 is common, with more ligase and longer incubation. Very large inserts sometimes clone better nearer 1:1–2:1 (their ends are scarce and precious), and screening a ratio ladder (1:1, 3:1, 5:1 in parallel) is cheaper than repeating a failed transformation.

Why not just use equal nanograms?

Because ligase counts molecules, not mass. Equal ng of a 1.5 kb insert and a 5 kb vector is actually a 3.3:1 molar ratio in the insert’s favor by accident — sometimes fine, often not the ratio you intended, and reversed (starved) when the insert is the larger fragment. The size term in the formula is the entire point: it converts your INTENDED ratio into mass honestly for any size pairing.

How much total DNA should a ligation have?

Typically 10–200 ng total in a 10–20 µL reaction for plasmid cloning. More is not better: high DNA concentration drives intermolecular joining (concatemers, multiple inserts) over the desired single-insert circles; very low concentration starves the bimolecular step. The defaults land at 190 ng total — the productive middle for a standard 10 µL setup scaled appropriately.

What does “fmol” mean in the result?

Femtomoles — 10⁻¹⁵ mol, the natural counting unit for DNA fragments (dsDNA ≈ 660 g/mol per bp, so 1 fmol of a 5 kb plasmid ≈ 3.3 ng). Reporting both species in fmol exposes the true ratio the reaction experiences, independent of fragment sizes. Vector products are often specified in fmol for exactly this reason.

Why did my ligation give only vector colonies?

The usual suspects, in order: incomplete vector digestion (uncut/partially cut vector transforms beautifully), dephosphorylation skipped or insufficient, insert ends damaged (UV overexposure during gel excision is a classic), dead ligase/buffer ATP — and only then, ratio error. The vector-only control ligation separates digestion problems from insert problems in one plate; run it every time.

Can this be used for Gibson assembly?

The mole math transfers: Gibson protocols want roughly equimolar fragments (ratio 1) with overlaps, total ~0.02–0.5 pmol scaled to fragment count. Set the ratio to 1 and apply the formula pairwise against your backbone; the ng answers remain valid. Golden Gate similarly leans on equimolarity per fragment — the size correction still does the work.

Is this ligation calculator free?

Yes — free, instant, no sign-up. Enter vector ng and length, insert length and desired molar ratio for insert nanograms with a femtomole audit.