1. What is the Ligation Calculator?

The Ligation Calculator determines the optimal mass of DNA insert needed for a ligation reaction based on the vector mass, lengths of both molecules, and desired molar ratio. This ensures efficient cloning with proper stoichiometry.

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)
• \( R \) — Desired molar ratio (insert:vector)

Explanation: The equation calculates the mass of insert DNA needed to achieve a specific molar ratio with the vector DNA, accounting for their relative lengths.

3. Importance of Proper Ligation Ratios

Details: Using the correct insert:vector 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 ratio is set to 3:1.

5. Frequently Asked Questions (FAQ)

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

Q2: How do I measure DNA concentrations?
A: Use a spectrophotometer (Nanodrop) or fluorometer (Qubit) for accurate DNA quantification.

Q3: What if my insert is much smaller than my vector?
A: The calculator automatically accounts for size differences - smaller inserts will require less mass to achieve the same molar ratio.

Q4: Can I use this for blunt-end ligations?
A: Yes, but blunt-end ligations typically require higher insert:vector ratios (5:1 to 10:1) due to lower efficiency.

Q5: What about dephosphorylated vectors?
A: When using dephosphorylated vectors, you may need to increase the insert:vector ratio to compensate for reduced ligation efficiency.