🔗 Ligation Calculator
Determine the insert mass and volume required to achieve a specific insert-to-vector molar ratio for cloning reactions.
Volume of vector DNA you plan to add to the ligation reaction.
Common ratios are 1:1, 3:1, or 5:1 insert to vector.
How to Use This Calculator
Input DNA characteristics
Provide the lengths (in base pairs) and concentrations (ng/µL) of your vector and insert DNA.
Select vector amount and ratio
Choose how much vector DNA you will add and the desired insert-to-vector molar ratio.
Prepare ligation mix
Use the calculated insert mass and volume to assemble your ligation reaction with ligase and buffer.
Formula
pmol = (ng × 1000) ÷ (bp × 650)
DNA molecular weight is approximated as 650 g/mol per base pair. To reach a target molar ratio (R):
Insert pmol = Vector pmol × R
Convert pmoles back to mass and volume using the insert length and concentration respectively.
Full Description
Successful ligation reactions depend on balancing insert and vector DNA to maximize cohesive-end annealing while minimizing undesired vector self-ligation. Typical ratios range from 1:1 for blunt ligations to 3:1 for sticky-end cloning.
By converting between mass and molar quantities, this calculator streamlines reaction setup and helps avoid common pitfalls such as adding too much insert DNA or exceeding the reaction volume.
Frequently Asked Questions
Can I change vector volume after calculating?
Yes. Adjust the vector volume input and recalculate. The insert requirements update automatically to maintain the selected molar ratio.
What reaction volume should I target?
Standard ligations run in 10–20 µL using 1× ligase buffer. Ensure the sum of DNA volumes plus buffer fits within your planned volume.
How accurate is the 650 g/mol per bp estimate?
It is a reliable average for dsDNA. For highly accurate molarity (e.g., oligos), use sequence-specific molecular weights.
Do I need to adjust for overhangs?
Overhang bases minimally affect molecular weight. Focus instead on ensuring compatible cohesive ends and proper phosphatase or kinase treatment where required.