Biology and life-science lab
Ligation calculator (insert : vector molar ratio)
Insert mass for a chosen insert:vector molar ratio, with picomoles of each fragment and pipetting volumes, for cloning ligations.
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Result
- Insert for 3.00 : 1
- 50.0 ng
- Vector
- 27.0 fmol
- Insert
- 80.9 fmol
Working
- insert ng = ratio × vector ng × insert bp ÷ vector bp = 3 × 50 × 1000 ÷ 3000 = 50.0 ng
- pmol = ng × 1000 ÷ (bp × 617.96 + 36.04)
| Insert : vector | Insert (ng) | Insert (fmol) |
|---|---|---|
| 1 : 1 | 16.7 | 27.0 |
| 2 : 1 | 33.3 | 53.9 |
| 3 : 1 | 50.0 | 80.9 |
| 5 : 1 | 83.3 | 135 |
| 7 : 1 | 117 | 189 |
| 10 : 1 | 167 | 270 |
- 3:1 is a common starting point for a single insert; small inserts (under about 200 bp) often need 5:1 to 10:1. Keep the total DNA in a 20 µL ligation to roughly 10–100 ng of vector.
- Moles use the NEB double-stranded DNA formula (5′-phosphorylated ends), which agrees with the length-ratio rule to within 0.1 %.
Method and limits
Enter the lengths of the linearised vector and the insert, the amount of vector and the molar ratio you want. Add concentrations to get pipetting volumes.
insert (ng) = ratio × vector (ng) × insert length (bp) ÷ vector length (bp)
pmol of dsDNA = ng × 1000 ÷ (bp × 617.96 + 36.04)
Equal numbers of molecules weigh in proportion to their lengths, so the molar ratio becomes a mass ratio scaled by the length ratio. Picomoles use the average mass of a base pair (617.96 g/mol) plus 36.04 g/mol for two 5′-phosphorylated ends, as in NEB’s calculator.
Limits
- 3:1 is a common start for one insert; very short inserts often need 5:1 to 10:1, and blunt or multi-fragment assemblies need their own optimisation.
- The concentrations must be accurate: measure purified fragments, ideally with a dye-based assay rather than A260 alone.
- Keep the total DNA in a typical 20 µL ligation to roughly 10–100 ng of vector, as the enzyme maker recommends.
Worked example
A 3000 bp vector (50 ng) and a 1000 bp insert at 3:1: insert = 3 × 50 ng × 1000 ÷ 3000 = 50.0 ng. The vector is 27.0 fmol and the insert 80.9 fmol.
References
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