1. What is the Ligation Calculator?

The Ligation Calculator helps determine the appropriate mass of DNA insert needed for a ligation reaction based on the vector mass, lengths of both molecules, and desired molar ratio. This ensures optimal conditions for successful cloning.

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

Details: The molar ratio between insert and vector is crucial for efficient ligation. A ratio of 3:1 (insert:vector) is commonly used, but may vary 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. The default molar ratio is set to 3.

5. Frequently Asked Questions (FAQ)

Q1: What is the typical molar ratio for ligation?
A: A 3:1 insert:vector ratio is standard, but some protocols may use 1:1 to 10:1 depending on the application.

Q2: How do I measure DNA concentration?
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 in the calculation.

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).

Q5: How does this compare to commercial ligation calculators?
A: This provides the same core functionality as commercial calculators, with the same underlying formula.