What this calculator does
Ligation is set up by molar ratio rather than by mass, because what matters is how many insert molecules are available per vector molecule. Since the two fragments are usually very different lengths, equal masses give wildly unequal molar amounts.
The correction is the length ratio. With a 3 kb vector and a 1 kb insert, a 1:1 molar ratio needs only a third as much insert by mass, because each insert molecule weighs a third as much. The commonly used 3:1 insert-to-vector ratio then works out at equal masses in this particular case, which is a coincidence of these lengths rather than a general rule.
The formula
The insert mass needed is the vector mass multiplied by the insert length divided by the vector length, multiplied by the desired molar ratio. The relative moles line shows the resulting molar amounts in arbitrary units so the ratio can be checked directly, which is a useful guard against an inverted length ratio.
| Term | Meaning |
|---|---|
| Molar ratio | The ratio of insert molecules to vector molecules, commonly 3:1 for a standard cloning ligation. |
| Insert | The fragment being cloned into the vector. |
| Vector | The plasmid backbone receiving the insert. |
| Length correction | Multiplying by insert length over vector length, which converts a molar ratio into a mass. |
The inputs explained
| Field | What to enter |
|---|---|
| Vector mass used (ng) | Vector mass in nanograms, typically 50 to 100 ng for a standard reaction. |
| Vector length (kb) | Vector length in kilobases. |
| Insert length (kb) | Insert length in kilobases. |
| Target molar ratio (insert:vector) | The desired insert-to-vector molar ratio. 3:1 is the usual starting point, with higher ratios sometimes helping for small inserts. |
When to use it
Setting up a standard cloning ligation
The routine case: a known vector mass and two known lengths, giving the insert mass to add.
Troubleshooting low colony counts
Too little insert gives mostly re-ligated empty vector. Raising the ratio is a standard first adjustment, and the calculation shows what mass that means.
Cloning a very small or very large insert
The length correction matters most at the extremes. A 200 bp insert into a 5 kb vector needs a tiny mass for a 3:1 ratio, small enough that pipetting accuracy becomes the limiting factor.
Worked examples
Every figure in the tables below is produced by this page’s own calculator at build time, so the numbers and the tool always agree. Select any row to load that scenario.
How much insert does each molar ratio need?
A fixed vector mass and fragment lengths across a range of molar ratios.
| Insert:vector molar ratio | Insert mass needed | Vector mass used | Relative moles (vector : insert, arbitrary units) |
|---|---|---|---|
| 1:1 | 16.67 ng | 50.00 ng | 16.667 : 16.667 |
| 3:1 | 50.00 ng | 50.00 ng | 16.667 : 50.000 |
| 5:1 | 83.33 ng | 50.00 ng | 16.667 : 83.333 |
| 10:1 | 166.67 ng | 50.00 ng | 16.667 : 166.667 |
Questions
What insert-to-vector ratio should I use?
3:1 is the standard starting point for a straightforward ligation. Higher ratios up to 10:1 can help with small inserts or difficult ligations, and 1:1 is sometimes used for very large inserts. If the first attempt gives few colonies, adjusting the ratio is among the first things to try.
Why not just use equal masses?
Because equal masses give very unequal molar amounts whenever the fragments differ in length. A 1 kb insert has three times as many molecules per nanogram as a 3 kb vector, so equal masses would give a 3:1 molar ratio by accident in that case and something quite different in any other.
Does vector length include the insert?
No. Use the length of the linearised vector backbone alone, without the insert. Including it overstates the vector length and consequently overstates the insert mass required.
What if the calculated insert mass is very small?
Pipetting accuracy becomes the constraint below about 1 µL of a dilute stock. Either dilute the insert further so a larger volume can be measured, or scale the whole reaction up by increasing the vector mass, which raises both figures proportionally.
For the concentrations these masses come from, see the DNA concentration calculator. For copy numbers rather than masses, see the DNA copy number calculator.