Lab-Tacular

Ligation Calculator

Works out what goes in a ligation reaction from the molar ratio you want.Enter the lengths and concentrations of the vector and the insert.

One per line: name ⇥ length (bp) ⇥ concentration (ng/µL) ⇥ molar ratio (optional). Without the fourth cell the default for the chosen assembly is used.

How to use it

  1. Enter the vector's length and concentration, and how much of it goes into the reaction.
  2. Choose the assembly. One insert defaults to 3 : 1; several fragments default to equimolar.
  3. Paste the inserts, one per line. Add a fourth cell to give a line its own molar ratio.
  4. Check the reaction volume, buffer strength and ligase volume. The defaults are 20 µL, 10× and 1 µL.
  5. Pipette in the order of the table. Water is the largest volume so it goes first, and the ligase goes last.

The formulas, and what to watch for

Matching the molar ratio is one division. The rest is filling the tube.

Mass to moles

A base pair has an average mass, so a length and a mass give an amount. The same average is used as in this site's ng ↔ pmol converter.

pmol
ng × 1000 / (bp × 650)
mass of 1 pmol
bp × 650 / 1000 ng

How much insert

The insert's amount is the vector's amount times the molar ratio, converted back into a mass and then a volume.

insert pmol
vector pmol × ratio
insert ng
insert pmol × length × 650 / 1000
insert µL
insert ng ÷ concentration

Filling the tube

The buffer volume is derived rather than looked up: a 10× buffer becomes 1× at one part in ten.

buffer µL
reaction volume ÷ buffer strength
water µL
reaction − DNA − buffer − ligase

What to watch for

  • When the water comes out negative nothing is shown but the size of the overshoot. It happens with dilute DNA, and putting in less vector is the easiest fix.
  • 3 : 1 is the starting point for standard cloning where the insert is smaller than the vector (Addgene). Large inserts and blunt ends usually mean trying a range of ratios in parallel.
  • Multi-fragment assemblies start equimolar. Addgene notes that some labs see a sharp drop in success beyond five fragments in one reaction.
  • 650 g/mol per base pair is an average. Short fragments depart from it, and that error goes straight into the molar ratio.
  • Add the ligase last and do not vortex. It is in glycerol and will layer; mix gently with the pipette.
  • This calculates what goes in, and stops there. Temperature, time and blunt-end handling come from your enzyme's manual.

Frequently asked questions

References

Where the reaction layout and the defaults come from. The ng ↔ pmol arithmetic is a definition, and the average molecular weight per base pair is imported from the ng ↔ pmol converter.

  1. Addgene. Protocol — DNA Ligation (How to Ligate Plasmid DNA). Read 2026-08-21.https://www.addgene.org/protocols/dna-ligation/The 10 µL layout (1 µL of 10× buffer or 2 µL of 5×, 0.5–1 µL of T4 ligase, water to 10 µL), the 3 : 1 insert : vector ratio for standard cloning, and about 100 ng of total DNA
  2. Addgene. Protocol — Gibson Assembly. Read 2026-08-21.https://www.addgene.org/protocols/gibson-assembly/The equimolar default for multi-fragment assemblies ("Yields will be best when the DNA fragments are present in equimolar concentrations") and the warning about a sharp drop in success beyond five fragments

NEB's protocol pages would not open when these values were transcribed (a bot check) and were not used. No Golden Gate–specific document was obtained, so the equimolar default rests on the Gibson document alone.

Often used together

Open →

ng ↔ pmol Converter

Converts between ng, pmol and copy number from nucleic acid length.

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Transformation Efficiency

Calculates transformation efficiency in cfu/µg from colony counts.

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DNA/RNA Concentration Calculator (A260)

Calculates nucleic acid concentration and purity ratios from A260.