1. What is the Insilico Ligation Model?

The Insilico Ligation Model calculates the required mass of DNA insert needed for a ligation reaction based on the vector mass, lengths of both molecules, and desired molar ratio. This helps optimize cloning efficiency.

2. How Does the Calculator Work?

The calculator uses the insilico ligation equation:

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 needed to achieve a specific molar ratio with the vector, accounting for their different lengths.

3. Importance of Molar Ratios in Ligation

Details: Using the correct insert:vector ratio is crucial for successful cloning. Typical ratios range from 3:1 to 10:1 (insert:vector) to favor insert-vector ligation over vector-vector self-ligation.

4. Using the Calculator

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

5. Frequently Asked Questions (FAQ)

Q1: What molar ratio should I use?
A: For standard cloning, 3:1 is common. For difficult ligations or large inserts, higher ratios (5:1 to 10:1) may help.

Q2: How do I measure vector mass accurately?
A: Use nanodrop or Qubit quantification after gel purification or column cleanup for most accurate results.

Q3: Does insert length affect ligation efficiency?
A: Yes, longer inserts typically ligate less efficiently and may require higher ratios or specialized enzymes.

Q4: Should I adjust for different DNA forms?
A: The calculator assumes linear molecules. For supercoiled vectors, you may need slightly more insert (10-20%).

Q5: What if my vector is very small/large?
A: The equation accounts for length differences automatically through the LV term.