Insert length
kb
Vector length
kb
Vector mass
ng
Insert / vector ratio
3:1
Insert mass
ng

Ligation Calculator

By Łucja Zaborowska

Our ligation calculator allows you to compute the optimal vector and insert mass required for a cloning reaction.

Are you not yet familiar with these topics and terms? In the article below we'll cover all the basic information you need. We'll also talk about vector ligation reactions and explain how to use the cloning molar ratio calculator and the vector ligation formula.

Our vector insert calculator works both ways - just input all the data you have and enjoy your results, whatever it is you're looking for 🔗

How to use the ligation calculator?

If you were looking for the neb ligation calculator - you've just found something better. You're just a few simple steps away from your solution:

  1. Enter your insert length - in either base pairs (bp) or kilobases (kb).
  2. Enter your vector length - in either base pairs (bp) or kilobases (kb).
💡 Convert these two values is not too tricky:
1000 bp = 1 kb
1 bp = 0.001 kb

  1. Enter your vector mass - in either nanograms (ng) or micrograms (μg).

  2. Choose the insert/vector molar ratio. The ideal ratio is 3:1. A larger amount of insert increases your chances of a successful cloning reaction.

  3. In this particular situation, your result will be the insert mass needed for the reaction. We recommend that you add at least 50 ng of insert.

How to calculate the molar ratio?

Our ligation molar ratio calculator uses the following equation:

Required insert mass = (Vector mass * Insert length)/Vector length * Molar ratio

where:

  • Required insert mass is given in nanograms (ng);
  • Vector mass is given in nanograms (ng);
  • Insert length is given in kilobases (kb);
  • Vector length is given in kilobases (kb); and
  • Molar ratio is that for insert/ vector.

Ligation reaction

The ligation aims to create a recombinant plasmid. In order to do that, we'll need to covalently connect two sticky ends - the one from the DNA backbone and the other from the DNA insert. This connection process is catalyzed by an enzyme - T4 DNA ligase - ATP is used as the source of energy.

What are the sticky ends?

Most restriction (scissor) enzymes cut the DNA asymmetrically. This process results in uneven ends; later, we may join them to compatible tips.

What is a vector?

A vector is a biological molecule that works as a carrier of a certain gene or expression (e.g. plasmids). We use it to implant the desired genetic material into a foreign cell.

Vectors in biology have a different definition to mathematical vectors!

Vector = Backbone + Insert

The backbone is the larger sequence that supports the vector.

What does the insert/vector molar ratio depend on?

The molar ratio depends on the size of each of its ingredients as well as their concentration. In most cases, when the vector's size is larger than the insert's size, a ratio 3:1 should be suitable.

Why do we need ligation?

  1. We use ligation in the molecular cloning of DNA. Bacteria with implanted plasmids multiply our desired DNA fragment:

    • We can use it for substance production (e.g. insulin);
    • They serve as disease models; and
    • They are tested as part of gene therapy.
  2. Ligation is an essential part of DNA replication

    • Ligation is what keeps us alive; and
    • Enzymes fix the DNA strain by ligation if it gets broken, protecting us from unwanted mutations and their effects on the body.
Łucja Zaborowska