Why use a molar ratio instead of equal masses?

Ligation efficiency depends on the number of DNA ends present, not their total mass. A short insert and a long vector at equal mass contain very different molar amounts, so matching ends requires correcting for fragment length. Working in a molar ratio keeps the chemistry balanced.

What insert:vector ratio should I use?

A 3:1 insert:vector molar ratio is a reliable default for sticky-end cloning. Blunt-end ligations often work better near 1:1 or 1:3, and difficult inserts can be pushed to 5:1. Running a small ratio series in parallel is the fastest way to find what works.

Which formula does this use?

Insert mass in ng equals the ratio times the vector mass times the insert length divided by the vector length. This works because the molar mass of double-stranded DNA is proportional to its length, so length cancels into the ratio of moles.

How are the pmol amounts calculated?

It uses an average of 650 g/mol per base pair of double-stranded DNA. Picomoles equal the mass in ng divided by length in bp times 650, scaled to picomole units. This is the same conversion used by most cloning kits.

Does this account for vector self-ligation?

No. The calculator only sets the insert:vector ratio. To suppress empty vectors, dephosphorylate the cut vector or use directional cloning with two different ends, and always run a vector-only no-insert control.

Ligation Molar Ratio Calculator (Insert:Vector)

Setting up a DNA ligation correctly means matching the number of insert and vector ends, not their masses. This calculator works out how many nanograms of insert to add for any insert:vector molar ratio, given the amount of vector you have and the sizes of both fragments.

How it works

Because the molar mass of double-stranded DNA is proportional to its length in base pairs, the number of moles in a sample is its mass divided by its length. To hit a target molar ratio R of insert to vector, the insert mass works out to:

insert ng = R x vector ng x (insert bp / vector bp)

The length terms convert the mass ratio into a true molar ratio. The tool also reports each fragment in picomoles using the standard conversion of about 650 g/mol per base pair:

pmol = ng / (bp x 650) x 1,000,000

Tips and example

Suppose you have 50 ng of a 5,000 bp vector and a 1,000 bp insert, and you want a 3:1 ratio. The insert mass is 3 x 50 x (1000 / 5000) = 30 ng. Note that the insert is one-fifth the length of the vector, so even at a 3:1 molar ratio you add less insert by mass than vector.

A few practical notes: keep total DNA in a 10-20 µL reaction modest, run a no-insert vector control on the same plate, and if you see few colonies, repeat at 1:1 and 5:1 before changing anything else. For blunt ends, lower ratios and longer incubations usually help.