Ligation Calculator

Calculate optimal insert:vector molar ratios for DNA ligation. Determine precise insert amounts for maximum cloning efficiency in restriction ligation and Gibson assembly.

Size of your plasmid backbone
Size of your DNA insert
Typical: 50-100 ng
Common ratios: 1, 3, or 5

Three-Way Ligation (Two Inserts)

Ratio for each insert relative to vector

Ligation with Volume Calculations

Measure with NanoDrop or Qubit
Measure with NanoDrop or Qubit

What is a Ligation Calculator?

A tool to calculate optimal insert:vector molar ratios for DNA ligation reactions. Proper ratios maximize transformation efficiency and colony yield in molecular cloning experiments.

How to Use This Calculator

Quick guide for ligation calculations:

  1. Enter your vector and insert sizes (bp)
  2. Input vector amount (typically 50-100 ng)
  3. Select molar ratio (3:1 recommended)
  4. View calculated insert amount needed
  5. Follow the ligation protocol provided

When to Use

Essential for molecular cloning:

  • Restriction enzyme cloning
  • Gibson assembly reactions
  • TA cloning
  • Blunt-end ligation
  • Three-way ligations
  • Golden Gate assembly

Calculation Formula

The molar ratio formula:

Insert (ng) = Vector (ng) × (Insert size / Vector size) × Ratio

Example: 50 ng vector (5 kb) with 1.5 kb insert at 3:1 ratio needs 45 ng insert.

Optimal Molar Ratios

Recommended ratios for different applications:

  • 3:1 - Standard for most ligations (recommended)
  • 1:1 - Large inserts (>5 kb) or blunt ends
  • 5:1 - Small inserts (<500 bp)
  • 10:1 - TA cloning or difficult ligations

Tips for Success

Maximize your ligation efficiency:

  • Use fresh, gel-purified DNA
  • Dephosphorylate vector with CIP/SAP
  • Keep total DNA <200 ng in 20 μL reaction
  • Use sticky ends when possible (better than blunt)
  • Include no-insert control (vector only)
  • Overnight ligation at 16°C gives best results

Troubleshooting

Q: No colonies after transformation?
A: Check vector dephosphorylation, verify insert orientation, try different ratios (1:1, 3:1, 5:1), ensure ligase is active.

Q: High background (empty vector)?
A: Improve vector dephosphorylation, gel-purify cut vector, reduce vector amount, use blue-white screening.

FAQ

Q: What's the difference between mass ratio and molar ratio?
A: Molar ratio accounts for DNA size, giving equal numbers of molecules. Mass ratio doesn't account for size and is less accurate.

Q: Can I use this for Gibson assembly?
A: Yes! Use 2-3× molar excess of inserts. Gibson assembly is more forgiving than restriction ligation.