1. What is the Ligation Calculator?

The Ligation Calculator helps determine the appropriate mass of DNA insert (MI) needed for a ligation reaction based on the vector mass (MV), their respective lengths, and the desired molar ratio (R).

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 formula 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: Using the correct insert:vector molar ratio is crucial for efficient ligation. A typical ratio ranges from 3:1 to 10:1 (insert:vector) depending on the application.

4. Using the Calculator

Tips: Enter vector mass in ng, insert and vector 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 most applications, a 3:1 insert:vector ratio works well. For TA cloning or blunt-end ligations, higher ratios (5:1 to 10:1) may be needed.

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

Q3: What if my insert is much smaller than my vector?
A: The calculator automatically accounts for size differences through the length parameters in the formula.

Q4: Can I use this for multiple fragment assemblies?
A: For multiple fragments, calculate each insert separately and combine them in the appropriate ratios.

Q5: What other factors affect ligation efficiency?
A: DNA ends (blunt vs sticky), DNA purity, phosphatase treatment, and ligase concentration all impact ligation success.