1. What is the Ligation Calculator?

The Ligation Calculator Insilico Plus helps determine the optimal mass of DNA insert needed for a ligation reaction based on vector mass, lengths of insert and vector, 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 your vector DNA in the ligation reaction.

3. Importance of Molar Ratio in Ligation

Details: Using the correct insert:vector molar ratio is crucial for efficient ligation. Typical ratios range from 1:1 to 10:1 (insert:vector), with 3:1 being commonly used.

4. Using the Calculator

Tips: Enter vector mass in ng, lengths in bp, and desired molar ratio. All values must be positive numbers. The default molar ratio is set to 3:1.

5. Frequently Asked Questions (FAQ)

Q1: What molar ratio should I use?
A: For standard cloning, 3:1 is recommended. For difficult ligations (large inserts or blunt ends), try 5:1 to 10:1.

Q2: How do I measure vector mass?
A: Use a spectrophotometer (Nanodrop) or fluorometer (Qubit) to quantify your linearized vector DNA.

Q3: What if my insert is a PCR product?
A: PCR products should be purified before ligation. Consider adding 15-30% more insert to account for potential inaccuracies in quantification.

Q4: Does this work for TA cloning?
A: Yes, but TA cloning typically requires more insert (5:1 to 10:1 ratio) due to lower efficiency.

Q5: What about blunt-end ligations?
A: Blunt-end ligations are less efficient. Use higher insert:vector ratios (5:1 to 10:1) and consider using more ligase or longer incubation times.