Cloning · Ligation setup
Ligation insert:vector ratio calculator.
Work out how many ng of insert to add for a chosen molar insert:vector ratio.
Inputs
Result
Enter your values.
How to calculate insert mass for a molar ratio
Ligation efficiency depends on the ratio of molecules, not mass, and a short insert weighs far less than the same number of vector molecules. With dsDNA at 660 g/mol per base pair: insert ng = ratio x vector ng x insert length / vector length. NEB's guidance is about 50 ng of vector (roughly 25 to 30 fmol depending on size), a 1:1 insert:vector ratio when the pieces are similar in size, and about 3:1 for a small insert such as 1 kb into a 3 kb vector.
Worked example. 50 ng of a 3,000 bp vector, a 1,000 bp insert, 3:1: 3 x 50 x 1,000 / 3,000 = 50 ng of insert. The vector is 50 ng / (3,000 x 660 g/mol) = 25.3 fmol and the insert 75.8 fmol.
Different kits and fragment types (very short inserts, blunt ends, multi-fragment assemblies) call for other ratios, so follow your enzyme's manual and try a few ratios. Convert any mass to copies with the DNA copy number calculator.
Frequently asked questions
What insert:vector ratio should I use?
NEB suggests 1:1 for similar-sized pieces and about 3:1 for a smaller insert, with higher ratios sometimes tried for very short inserts. Optimise for your system.
How much vector should I use?
About 50 ng is a typical amount for standard cloning according to NEB, but follow your kit's manual.
Why is the insert mass not equal to the vector mass?
Equal masses of different lengths contain different numbers of molecules. The formula corrects for length.
Does this work for sticky and blunt ends?
The mass-to-moles arithmetic is the same. Blunt-end ligation is less efficient and often uses different conditions.
Sources
Checked
- NEB, How do I calculate how much DNA to add to a ligation reaction (vector amount and molar ratios)
- Promega BioMath, DNA mass to pmol (660 pg per pmol of base pairs)
Runs entirely in your browser. Nothing you type is sent or stored.
Research use and scope
This is arithmetic for research-bench planning, not clinical, diagnostic or safety advice. Conversion factors are standard approximations; strain, instrument, reagent and protocol differences matter. Check results against your own protocol, kit manual and lab supervisor, and follow your institution's biosafety rules.