Single ligation
Classic restriction-ligation: one vector + one insert at your chosen insert:vector molar ratio.
Typical: 20–100 ng per 20 µL reaction.
Use the insert fragment length, not the whole plasmid.
Overrides the preset buttons. Blunt ends often use 5:1–10:1.
Fill both stocks to get pipetting volumes.
ng of insert needed
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Two inserts (double digest)
Three-fragment ligation: each insert is calculated independently against the same vector mass.
total insert mass (A + B)
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Vector-only control
The religation control: everything except the insert. Same vector nanograms, so any colonies here mean background religation — not successful cloning.
vector mass for control
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Reading the control: a clean vector-only plate (few or no colonies) plus colonies on the
ligation plate = your insert likely got in. A crowded control plate = religation background — check digest
completeness and dephosphorylation before blaming the ratio.
Reaction scale planner
Scale one validated reaction to multiple transformations, with 10% pipetting overage.
Copy from the Single ligation result above.
total DNA for all reactions (+10% overage)
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Mass ⇄ moles converter
Convert between ng, pmol and fmol for any dsDNA fragment (660 g/mol per bp).
result
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Boundary: need molecule counts (copies/µL via Avogadro)?
That is a different question — use our DNA Copy Number Calculator.
This tool answers mass stoichiometry for ligation reactions.