Ligation Calculator
Calculate optimal vector and insert mass ratios for DNA ligation and molecular cloning reactions
Calculate DNA Ligation Ratios
Length of DNA fragment to be inserted
Length of cloning vector (backbone)
Amount of vector DNA in ligation reaction
Recommended: 3:1 for optimal ligation efficiency
Ligation Results
Enter insert length, vector length, vector mass, and molar ratio to calculate
All fields are required for accurate ligation calculations
Ligation Efficiency Tips
Example Calculation
Typical Cloning Scenario
Insert: 1.5 kb gene of interest
Vector: 5.0 kb pUC19 plasmid
Vector amount: 100 ng
Desired ratio: 3:1 (insert:vector)
Calculation
Insert mass = (100 ng × 1.5 kb × 3) / 5.0 kb
Insert mass = (450) / 5.0
Insert mass = 90 ng
Total DNA = 190 ng
Ligation Setup
• Vector (digested): 100 ng
• Insert (digested): 90 ng
• T4 DNA ligase: 1 μl (400 U)
• T4 ligase buffer: 2 μl (10×)
• Water to 20 μl total volume
• Incubate: 16°C overnight or 25°C for 2 hours
Quick Reference
Optimal Ratio
Insert:Vector molar ratio
Best for most cloning reactions
Minimum Insert
50 ng recommended
For reliable ligation
Unit Conversion
1000 bp = 1 kb
1000 ng = 1 μg
Common Cloning Vectors
Tip: Check vector specifications for exact length and consider linearization efficiency.
Understanding DNA Ligation
What is DNA Ligation?
DNA ligation is the process of joining two DNA fragments together using T4 DNA ligase enzyme. This is a fundamental technique in molecular cloning used to create recombinant plasmids by inserting DNA fragments into vector backbones.
Key Components
- •Vector: DNA backbone (usually plasmid) that carries the insert
- •Insert: DNA fragment to be cloned (gene of interest)
- •T4 DNA Ligase: Enzyme that catalyzes phosphodiester bond formation
- •ATP: Energy source for the ligation reaction
Ligation Formula
Insert mass = (Vector mass × Insert length × Ratio) / Vector length
Molar ratio calculation formula
- Insert mass: Required DNA insert amount (ng)
- Vector mass: Amount of vector DNA (ng)
- Insert length: Size of insert in kb
- Vector length: Size of vector in kb
- Ratio: Insert:vector molar ratio (typically 3:1)
Molecular Weight: ~650 Da per base pair (average)
Ligation Mechanism
T4 DNA ligase catalyzes the formation of phosphodiester bonds between the 3'-hydroxyl and 5'-phosphate groups of adjacent DNA fragments. The reaction requires compatible cohesive ends (sticky ends) or can work with blunt ends at higher enzyme concentrations.
Restriction Digestion
Vector and insert cut with compatible enzymes
Creates sticky or blunt ends
Dephosphorylation prevents self-ligation
Ligation Reaction
T4 ligase joins compatible ends
ATP provides energy for bond formation
Optimal temperature: 16°C overnight
Transformation
Recombinant plasmids enter bacteria
Selection using antibiotic resistance
Colony screening confirms insert
Optimization Strategies
Molar Ratio Optimization
- • 1:1 ratio - minimal background, lower efficiency
- • 3:1 ratio - optimal for most applications
- • 5:1 ratio - maximum efficiency, more background
- • >10:1 ratio - excessive insert, wasteful
Reaction Conditions
- • Temperature: 16°C (overnight) or 25°C (2h)
- • Buffer: T4 ligase buffer with ATP
- • Volume: 10-20 μl total reaction
- • Controls: vector-only and no-ligase