Ligation Calculator
Calculate the exact mass of insert DNA needed for any cloning ligation reaction. Enter insert and vector lengths, vector mass and molar ratio to get the required insert mass in nanograms instantly.
Ligation Calculator Tool
Ligation calculator with minimum insert warning and total DNA mass
Most ligation calculators return only the insert mass. This tool also shows total DNA in the reaction, warns when insert falls below the 50 ng recommended minimum, and provides a ratio guide for sticky-end, blunt-end and Gibson Assembly cloning.
How to calculate insert mass for ligation
Ligation conditions by end type
| End type | Recommended ratio | Efficiency | Conditions |
|---|---|---|---|
| Sticky (4-base overhang) | 3:1 | High | 16 C / 16 h or 25 C / 1 h |
| Blunt end | 5–7:1 | Low | 16 C / 16 h + PEG 4000 |
| TA cloning | 3:1 | High | Room temperature / 5 min |
| Gibson Assembly | 1:1 | Very high | 50 C / 15 min |
LazyTools Ligation Calculator vs the competition
| Feature | LazyTools | Omni | NEB Ligation | Addgene |
|---|---|---|---|---|
| Insert mass calculation | ✓ Yes | ✓ Yes | ✓ Yes | ✗ Guide only |
| All 5 molar ratios (1:1-7:1) | ✓ Yes | ✓ Yes | ~ 3 ratios | ✗ No |
| 50 ng minimum warning | ✓ Yes | ✗ No | ✗ No | ✗ No |
| Total reaction DNA shown | ✓ Yes | ✗ No | ✗ No | ✗ No |
| Blunt-end ratio guidance | ✓ Yes | ✗ No | ~ Partial | ✓ Yes |
| Gibson Assembly note | ✓ Yes | ✗ No | ✗ No | ~ Partial |
| No login required | ✓ Yes | ✓ Yes | ✓ Yes | ✓ Yes |
| 100% browser-side | ✓ Yes | ✗ No | ✗ No | ✗ No |
DNA Ligation Calculator — A Complete Guide
DNA ligation joins a gene of interest (insert) into a circular carrier (vector/plasmid), creating a recombinant plasmid that can be transformed into bacteria for amplification. Getting the insert-to-vector molar ratio right is critical for efficient cloning.
How to calculate insert mass for ligation
The formula is: Insert mass (ng) = Vector mass (ng) x (Insert bp / Vector bp) x Molar ratio. This converts molar ratio into mass terms, accounting for the fact that a shorter insert has a higher molar concentration per gram. Example: 100 ng of a 4500 bp vector with a 1200 bp insert at 3:1 ratio requires 100 x (1200/4500) x 3 = 80 ng of insert.
Choosing the correct insert-to-vector molar ratio
For sticky-end (cohesive-end) ligation, 3:1 is standard. Restriction enzymes generating 4-base overhangs create complementary ends that hybridise before ligation, dramatically increasing efficiency. For blunt-end ligation, efficiency is 10-100x lower; use 5:1 to 7:1 plus PEG 4000 as a crowding agent. For Gibson Assembly and Golden Gate, the T4 formula does not apply — use 1:1 for Gibson.
Why the 50 ng insert minimum matters
Using at least 50 ng of insert ensures sufficient molar excess to compensate for ligation efficiency losses, pipetting variability and competition between insert-vector and vector self-ligation. Below this, recombinant colony counts drop substantially. To prevent vector self-ligation, treat linearised vector with calf intestinal phosphatase (CIP) to remove 5' phosphate groups before ligation.