Sticky-end ligation at 3:1
Set up a standard cloning reaction with a 1 kb insert.
Ligation Calculator
FigranEnter fragment lengths and concentrations to get the reaction setup in µL.
- Insert:vector ratios
- Gibson, HiFi and In-Fusion
- Copy or print the setup
3:1 ligation · pUC19 + Insert · 20 µL
Insert 55.85 ng · 2.23 µL| Component | 3:1 |
|---|---|
| Water | 13.77 µL |
| 10× T4 DNA ligase buffer | 2.00 µL |
| pUC19 | 1.00 µL |
| Insert | 2.23 µL |
| T4 DNA ligase (add last) | 1.00 µL |
| Total | 20.00 µL |
pUC19: 50.00 ng · 0.0301 pmol · Insert: 55.85 ng · 0.0904 pmol
16 °C overnight or room temperature 10 min · heat-inactivate 65 °C 10 min
Prepared by ____________________ Date __________
Ligation calculator examples
Start with a reaction and adapt it for your fragments.
NEB protocol example: 4 kb vector, 1 kb insert
Reproduce the T4 DNA ligase protocol example.
TA cloning into pGEM-T Easy
Calculate a 10 µL ligation with a PCR product.
Large insert into a small vector
Use equal molar amounts for a larger insert.
NEBuilder HiFi assembly with a 150 bp fragment
Assemble a vector, GFP and a short linker.
In-Fusion cloning with three inserts
Calculate a 200 ng assembly with three inserts.
Blunt-end ratio series with a vector-only control
Compare three ratios and a background control.
Five-fragment Gibson Assembly
Fit five equimolar fragments into the available DNA volume.
How to set up a ligation reaction
Plan your reaction in four steps.
1Enter fragment data
Enter the length and concentration of the vector and insert.
2Choose a reaction
Select T4 DNA ligase or an assembly kit.
3Set the ratio
Choose an insert:vector ratio or a series with a control.
4Take the setup to the bench
Copy or print the reaction table and add the components in order.
Insert to vector ratio
Choose a starting ratio for your cloning method.
| Method | Insert:vector | Total DNA | Incubation |
|---|---|---|---|
| T4 sticky ends | 3:1 | 50 ng vector | 16 °C overnight / RT 10 min |
| T4 blunt ends | 3:1–5:1 | 50 ng vector | 16 °C overnight / RT 2 h |
| TA cloning | 3:1 | 50 ng vector | Room temperature 1 h or 4 °C overnight |
| Large insert | 1:1 / 1:3 | 50 ng vector | Follow the selected protocol |
| NEBuilder HiFi | 2–3 fragments: 2:14–6 fragments: 1:1Fragments under 200 bp: 5:1 | 2–3 fragments: 0.03–0.2 pmol4–6 fragments: 0.2–0.5 pmol | 2–3 fragments: 50 °C, 15 min4–6 fragments: 50 °C, 60 min |
| Gibson Assembly | 2–3 fragments: 2:14–6 fragments: 1:1Fragments under 200 bp: 5:1 | 2–3 fragments: 0.02–0.5 pmol4–6 fragments: 0.2–1.0 pmol | 2–3 fragments: 50 °C, 15 min4–6 fragments: 50 °C, 60 min |
| In-Fusion | 50–350 bp: 5:1; otherwise 2:1 | 200 ng | 50 °C 15 min |
The insert:vector ratio is a molar ratio, not a mass ratio.
High ratios above 10:1 may favor multiple inserts.
References: NEB T4 DNA Ligase · NEBuilder HiFi · Gibson Assembly · Takara In-Fusion Snap Assembly manual (071320), pp. 7–8
Ligation calculation formula
Convert the molar ratio into mass and volume.
3 × 50 ng × 1,000 / 2,686 = 55.85 ng; 55.85 ng / 25 ng/µL = 2.23 µL.
Ligation calculator FAQ
What insert to vector molar ratio should I use for ligation?
Start with 3:1 for sticky ends and test 1:1, 3:1 and 5:1 if needed. For blunt ends, 3:1–5:1 is a useful starting range; a large insert may work better at 1:1 or 1:3. These are molar ratios.
How do I calculate how much insert to add?
Multiply vector mass by the insert:vector ratio and the insert-to-vector length ratio. For 50 ng of a 2,686 bp vector with a 1,000 bp insert at 3:1, use 55.85 ng, or 2.23 µL at 25 ng/µL.
How do I convert ng to pmol for double-stranded DNA?
Divide ng × 1,000 by bp × 617.96 + 36.04. Reverse the equation to convert pmol to ng; this uses an average double-stranded DNA molecular weight.
What if the DNA volume is too large or too small to pipette?
Reduce the vector mass or concentrate the DNA if it will not fit. For volumes below 0.5 µL, dilute the stock first. For NEB assemblies, unpurified PCR fragments should occupy no more than 20% of the reaction.
How much DNA goes into a Gibson or HiFi assembly?
For 2–3 fragments, NEB recommends 0.02–0.5 pmol for Gibson or 0.03–0.2 pmol for HiFi. For 4–6 fragments, the ranges are 0.2–1.0 pmol and 0.2–0.5 pmol respectively.
What ratio should I use for small fragments or 4–6 fragment assemblies, and how long should overlaps be?
For NEB HiFi and Gibson assemblies, use a 5-fold excess for fragments under 200 bp and equal molar amounts for 4–6 fragments. NEB suggests 15–20 bp overlaps for 2–3 fragments and 20–30 bp for 4–6 fragments. For In-Fusion, use 5–15:1 for 50–150 bp and 3–5:1 for 150–350 bp fragments; 5:1 is a starting point for both ranges. Check overlap primers with the Primer Tm Calculator.
Do I need a vector-only control?
A vector-only control helps reveal background from self-ligation or uncut vector. Select Vector-only control to add a separate reaction.
Do the results match NEBioCalculator, Promega and Takara calculators?
The same length-ratio calculation gives 55.85 ng for the default example and 18.62 ng at 1:1. NEBuilder Protocol Calculator and the Takara calculator also agree with the corresponding assembly examples at their displayed precision.
Can I get fragment lengths from a sequence file?
Paste a sequence or FASTA text into Length to count its bases. You can also enter a length from a GenBank or SnapGene file. Draw the assembled construct with Plasmid Map Maker.