1. What is the Ligation Calculator?

The Ligation Calculator Insilico Price determines the optimal mass of insert DNA needed for a ligation reaction based on vector mass, lengths of both molecules, and desired molar ratio.

2. How Does the Calculator Work?

The calculator uses the ligation formula:

Where:

• \( MI \) — Insert mass (ng)
• \( MV \) — Vector mass (ng)
• \( LI \) — Insert length (bp)
• \( LV \) — Vector length (bp)
• \( R \) — Molar ratio (insert:vector)

Explanation: The equation calculates the mass of insert DNA needed to achieve a specific molar ratio with the vector DNA in the ligation reaction.

3. Importance of Molar Ratio in Ligation

Details: Proper molar ratios are crucial for efficient ligation. Typical ratios range from 1:1 to 10:1 (insert:vector) depending on application.

4. Using the Calculator

Tips: Enter vector mass in ng, lengths in bp, and desired molar ratio. All values must be positive numbers.

5. Frequently Asked Questions (FAQ)

Q1: What is a typical molar ratio for ligation?
A: For standard cloning, 3:1 is common. For TA cloning or blunt-end ligation, higher ratios (5:1 to 10:1) may be needed.

Q2: How do I measure vector mass?
A: Use a spectrophotometer (Nanodrop) or fluorometer (Qubit) after vector purification and concentration determination.

Q3: Does insert length affect ligation efficiency?
A: Yes, very short inserts (<50bp) or very long inserts (>10kb) may require optimization of conditions.

Q4: What if I get poor ligation results?
A: Consider testing different ratios, using fresh ATP, or optimizing incubation times/temperatures.

Q5: Can this be used for Gibson Assembly?
A: No, Gibson Assembly uses different stoichiometry. This calculator is for traditional restriction enzyme-based ligation.