If you have ever stared at a ligation reaction and wondered how many nanograms of insert make a 3:1 ratio, this is the calculator for you.
Ligase joins molecules, not grams. A short insert has far more copies per nanogram than a long vector, so you can’t just add equal masses and hope. This ligation calculator turns your vector amount, your two fragment sizes and the ratio you want into the mass of insert to add, plus the picomoles and the volumes to pipette.
How to use the Ligation Calculator#
- Enter the vector amount. Use the nanograms of cut vector going into the reaction. 50 ng is a common starting point.
- Enter both lengths. Give the vector backbone and the insert in base pairs, measured after digestion.
- Set the molar ratio. Enter the insert-to-vector ratio, for example 3 for 3:1.
- Add the concentrations (optional). Enter the stock concentrations in ng/µL to get the volumes to pipette.
- Read the result. You get the insert mass, the picomoles of each fragment and the two volumes.
The formula#
To keep the molar ratio, the mass of insert has to scale with the length of the two fragments:
To convert mass to moles, double-stranded DNA is taken as 650 g/mol per base pair:
A worked example#
You have 50 ng of a 3,000 bp vector and a 1,000 bp insert, and you want a 3:1 ratio.
- Insert mass: 3 × 50 × 1,000 ÷ 3,000 = 50 ng
- Vector in picomoles: 50 × 1,000 ÷ (3,000 × 650) = 0.0256 pmol
- Insert in picomoles: 50 × 1,000 ÷ (1,000 × 650) = 0.0769 pmol
0.0769 divided by 0.0256 is 3, so the ratio holds. If your vector stock is 25 ng/µL you pipette 2 µL, and if the insert is at 10 ng/µL you pipette 5 µL.
Try it with a smaller insert. A 500 bp insert with a 4,000 bp vector at 50 ng and 3:1 needs only 18.75 ng of insert. The shorter the insert, the less mass you need for the same number of molecules.
Which ratio should you pick?#
There’s no single correct ratio. These are common starting points, and your ligase manufacturer’s protocol is the final word:
| Situation | Typical starting ratio (insert : vector) |
|---|---|
| One insert with compatible sticky ends | 3:1 |
| Blunt-end ligation | Higher, often 5:1 or more |
| Very small inserts or adapters | Higher again |
| Troubleshooting a failed ligation | Try a range, such as 1:1, 3:1 and 10:1 |
Going very high isn’t free. Too much insert can lead to inserts joining each other, and that’s the sort of thing that leaves you with a plate of colonies and none of them right.
Get the lengths right#
The most common mistake is using the length of the whole plasmid. If your digest removes a stuffer or a small piece, the backbone you’re ligating into is shorter than the original plasmid. Use the size of the fragment that goes into the tube, which you can confirm on a gel. The same applies to the insert. Count the digested fragment, not the original PCR product.
Controls that save you a day#
Run a vector-only ligation alongside the real one. If that plate has lots of colonies, the vector is closing up on itself, and you may need to dephosphorylate it or cut it more completely. A no-ligase control is also useful for checking leftover uncut plasmid.
This tool is for planning the reaction. Reaction time, temperature and buffer depend on the enzyme you use, so follow its protocol.
Troubleshooting a ligation#
If you get no colonies, check that the insert and vector have compatible ends, that the ligase and buffer are fresh (the ATP in the buffer breaks down with repeated freezing and thawing) and that the DNA was not damaged by UV light during gel purification. If you get many colonies with no insert, the vector is probably re-ligating, so dephosphorylate it or use a double digest with two different enzymes. Run a vector-only ligation as a control. Small inserts under 100 bp often need higher ratios, and large inserts of several thousand bp often work better at lower ones.
Frequently asked questions#
Multiply the molar ratio by the vector mass and by the insert length, then divide by the vector length. That gives the insert mass in nanograms.
Blunt ends join less efficiently than sticky ends, so people often start higher than 3:1, such as 5:1 or above. Check your ligase protocol.
It’s the average molecular weight of one base pair of double-stranded DNA. It’s used to convert between mass and moles.
Many protocols suggest around 50 ng of vector, but the right amount depends on your enzyme and protocol.
No. The 650 g/mol figure assumes double-stranded DNA, so the mass-to-mole conversion would be wrong.
Many protocols use 16 °C overnight or room temperature for 10 minutes to an hour with a quick ligase buffer. Follow your enzyme supplier’s instructions. Dephosphorylating the vector reduces self-ligation, and a vector-only control tells you how much background to expect.