What is DNA ligation?
DNA ligation is a fundamental step in molecular cloning. It involves joining linear DNA fragments together with covalent bonds, typically linking an insert DNA fragment into a sequence-compatible plasmid vector. This is done using an enzyme called DNA ligase, such as T4 DNA Ligase.
Why is the molar ratio important?
For a successful ligation and subsequent transformation, you need the right balance of insert and vector molecules.
If you have too much vector, you increase the chance of the vector ligating to itself (empty vector background). If you have too much insert, multiple inserts might ligate into the same vector, or insert-insert concatemers can form. The most common and widely successful molar ratio of Insert to Vector is 3:1.
Ligation calculation formula
Because mass does not equal molarity (longer DNA fragments weigh more per mole than short ones), you must calculate the required insert mass based on the relative lengths of the DNA fragments. The formula is:
- Insert Mass: The amount of insert DNA needed (in ng)
- Insert Length: Base pairs (bp) or kilobases (kb) of the insert
- Vector Length: Base pairs (bp) or kilobases (kb) of the vector. (Make sure both lengths use the same unit!)
- Vector Mass: The amount of vector DNA you plan to use (in ng, typically 50-100 ng)
- Molar Ratio: Desired ratio of insert to vector (e.g., 3 for a 3:1 ratio)
How to use this calculator
Simply input the lengths of your vector and insert, the amount of vector you are putting into the reaction, and your desired ratio. We default the ratio to 3, but you can adjust it to 1, 5, or 10 depending on your cloning strategy (for example, blunt-end ligations often require higher ratios like 5:1 or 10:1).