Ligation Calculator

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How to Use the Ligation Calculator

1. 1. Enter the fragment sizes. The length of the vector backbone and of the insert, in base pairs. Use the cut and purified fragment's length, not the plasmid it came from.
2. 2. Set the vector amount and the ratio. 50 ng of vector is a common starting point. The ratio is the number of insert molecules per vector molecule: enter 3 for a 1:3 vector:insert ligation.
3. 3. Add the stock concentrations (optional). With the vector and insert in ng/µL, the calculator converts the masses into volumes and fills the rest of the tube with water.
4. 4. Read the recipe and the notes. Copy the table straight into your notebook. Any warning, such as DNA that is too dilute for efficient ligation, appears beneath it.

The Insert Mass Formula

Ligation works on molecules, not nanograms, so a short insert at the same mass as a long vector is present in far larger numbers. Converting a molar ratio to a mass only needs the two lengths, because a fragment's mass is proportional to its length:

This is the calculation behind New England Biolabs' protocol for T4 DNA Ligase (M0202), which pairs 50 ng of a 4 kb vector with 37.5 ng of a 1 kb insert for a 1:3 vector:insert ratio in a 20 µL reaction: 3 × 50 × 1,000 ÷ 4,000 = 37.5 ng. Because the formula uses only a length ratio, the insert mass never depends on the base-pair mass you choose. That choice affects only the fmol figures. NEB's published average of 617.96 g/mol per base pair gives NEB's 0.020 and 0.060 pmol for that example; the 660 g/mol convention used by many suppliers and by the DNA Concentration Calculator gives values about 7% lower.

Choosing a Ratio and an Amount

• Ratio. NEB describes vector:insert ratios from 1:1 to 1:10 as optimal for a single insertion, and up to 1:20 for short adaptors. 1:3 is the usual first attempt. If a ligation fails, try 1:1 and 1:5 side by side rather than guessing.
• Vector amount. NEB recommends 20–30 fmol of vector — about 50 ng for a 4 kb plasmid.
• Concentration. Keep the overall DNA concentration (vector plus insert) between 1 and 10 ng/µL. Below 1 ng/µL the two ends of the same molecule are more likely to find each other than an insert, which favours self-circularisation.
• Cohesive and blunt ends. NEB's protocol incubates cohesive ends at 16 °C overnight or at room temperature for 10 minutes, and blunt or single-base overhangs for 16 °C overnight or 2 hours at room temperature. Heat-inactivate at 65 °C for 10 minutes, and add the ligase last.
• Controls. A cut vector with compatible ends can recircularise on its own. A vector-only control, the same reaction with water instead of insert, shows how much background to expect.

Worked Example: A 1.2 kb Insert Into a 3 kb Vector

You have 100 ng of a 3,000 bp cut vector and want to insert a 1,200 bp fragment. For a 1:3 ratio, the insert mass is 3 × 100 × 1,200 ÷ 3,000 = 120 ng. For 1:5 it is 5 × 100 × 1,200 ÷ 3,000 = 200 ng. In moles, 100 ng of that vector is 53.94 fmol with NEB's base-pair mass, and 120 ng of the insert is 161.8 fmol — three times as many molecules, as intended. Note that 53.94 fmol is above NEB's 20–30 fmol guide: 50 ng of vector would be closer to it.

With the calculator's default inputs (4,000 bp vector at 50 ng, 1,000 bp insert, ratio 3, stocks at 100 and 50 ng/µL, 20 µL total, 1 µL of ligase), it gives 37.5 ng of insert and this recipe: 2 µL of 10X buffer, 0.5 µL of vector, 0.75 µL of insert, 15.75 µL of water and 1 µL of ligase. The DNA is at about 4.4 ng/µL, inside the 1–10 ng/µL window.

Troubleshooting a Failed Ligation

• Many colonies on the vector-only control. The vector is closing on itself. Dephosphorylate it, or cut it with two enzymes that leave incompatible ends.
• No colonies at all. Check the DNA quantity first: measure both fragments after purification, because gel extraction can lose a large share of the DNA. Then check that the buffer's ATP has not degraded through repeated freeze–thaw.
• Colonies but no insert. Raise the ratio, or check that restriction enzymes were fully inactivated and that the insert's ends are compatible with the vector.

Frequently Asked Questions

How do you calculate the amount of insert for a ligation?

Multiply the molar ratio by the vector mass and by the insert length, then divide by the vector length. For 50 ng of a 4,000 bp vector and a 1,000 bp insert at a 1:3 ratio, that is 3 × 50 × 1,000 ÷ 4,000 = 37.5 ng. The lengths must be in the same unit; base pairs are standard.

What is the best vector to insert ratio?

There is no single best value, but 1:3 is the standard starting point and NEB considers 1:1 to 1:10 optimal for a single insert. Use 1:1 when the insert is large and 1:5 to 1:10 when it is small or the DNA concentrations are uncertain. Running two ratios in parallel is cheaper than repeating a failed ligation.

How much DNA should I use in a ligation?

NEB recommends 20–30 fmol of vector, which is roughly 50 ng for a 4 kb vector, with the total DNA between 1 and 10 ng/µL in the finished reaction. If your reaction volume pushes the concentration below that range, use a smaller volume rather than diluting with water.

Does the ratio mean mass or moles?

Moles. A 1:3 vector:insert ratio means three insert molecules per vector molecule, which is why the insert's mass depends on its length relative to the vector. Equal masses of a 1 kb insert and a 4 kb vector would be a 4:1 insert excess, not 1:1.

How long should a ligation run?

For T4 DNA ligase, NEB's protocol gives 10 minutes at room temperature or overnight at 16 °C for cohesive ends, and 2 hours at room temperature or overnight at 16 °C for blunt ends or single-base overhangs. Heat inactivation at 65 °C for 10 minutes follows; transform 1–5 µL of the reaction.

Why do the fmol values here differ from another calculator?

The mass of a base pair is an average and sources disagree: 660 g/mol is the common convention, while NEB publishes 617.96 g/mol. The choice changes pmol and fmol by about 7% but not the insert mass, because that depends only on the ratio of the two lengths.