Insert mass follows the molar ratio and the length ratio
The molar ratio (insert:vector) sets how many insert molecules you want per vector molecule; the length ratio converts that molecule count into a mass, because a longer fragment weighs more. So a short insert needs less mass than a long one to hit the same molar ratio.
- Worked example. 100 ng vector (5000 bp), 2000 bp insert, 3:1 → 100 × (2000 ÷ 5000) × 3 = 120 ng insert.
- Rule of thumb. If the insert is about 40 percent of the vector length and you want 3:1, the insert mass works out to roughly 1.2 × the vector mass.
Molar ratio by end type
| End type | Insert:vector | Note |
|---|---|---|
| Cohesive (sticky) | 3:1 | The default for most cloning |
| Blunt | 5:1 to 10:1 | Lower efficiency; often add PEG |
| Single-fragment / self | 1:1 | Recircularization, low insert bias |
Aim for about 50 to 100 ng of vector per reaction. Too little DNA and fragments rarely meet; too much and you get concatemers instead of clean single-insert products.
Insert needed for 100 ng of common vectors at 3:1
| Vector | Size | 2000 bp insert |
|---|---|---|
| pUC19 | 2686 bp | 223 ng |
| pBR322 | 4361 bp | 137 ng |
| pBAD24 | 5385 bp | 111 ng |
| pET28 | 5550 bp | 108 ng |
Same insert, same ratio: a smaller vector demands more insert mass, because each vector molecule weighs less. Values in ng.
Common questions
How do I calculate the insert mass for a ligation?
Multiply the vector mass by the ratio of insert length to vector length, then by the molar ratio you want. For 100 ng of a 5000 bp vector, a 2000 bp insert at 3:1: 100 x (2000/5000) x 3 = 120 ng.
What molar ratio should I use?
Use 3:1 insert to vector for cohesive (sticky) ends, which covers most cloning. Blunt ends ligate less efficiently, so raise it to 5:1 or even 10:1. Start at 3:1 and increase only if you get few colonies.
Why does fragment length matter?
The molar ratio is about the number of molecules, not their mass. A longer fragment weighs more per molecule, so to keep the same 3:1 count of insert molecules you scale the mass by the insert-to-vector length ratio.
