Ligation Calculator

ng
bp
bp
Typically 3:1 for standard cloning; use 5–7:1 for small inserts.
Biology

Calculate the amount of insert DNA needed for a ligation reaction based on vector amount, sizes, and desired molar ratio.

Formula

Insert (ng) = Vector (ng) × (Insert bp ÷ Vector bp) × Molar Ratio
  • Standard molar ratio for blunt-end ligation: 5–10:1. Sticky ends: 3:1.
  • Larger inserts require more ng of DNA per mole.
  • Use fresh vector linearized with restriction enzymes for best results.

50 ng vector (3000 bp), 1000 bp insert, 3:1 ratio

Inputs
  • Vector DNA Mass: 50 ng
  • Vector Size: 3000 bp
  • Insert Size: 1000 bp
  • Insert:Vector Molar Ratio: 3

Insert = 50 × (1000/3000) × 3 = 50 ng.

Frequently asked questions

What molar ratio should I use?
3:1 for sticky ends; 5:1 to 10:1 for blunt ends or small inserts.
What concentration should the reaction be?
Total DNA should be 1–10 ng/µL in the reaction; total volume typically 10–20 µL.