1. What is the Ligation Calculator?

The Ligation Calculator Insilico 2 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.

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: 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.

5. Frequently Asked Questions (FAQ)

Q1: What is a typical molar ratio for ligation?
A: A 3:1 insert:vector ratio is commonly used, but optimal ratios may vary depending on the specific application.

Q2: How do I measure vector mass?
A: Vector mass can be determined using a spectrophotometer (Nanodrop) or fluorometric methods like Qubit.

Q3: What if my insert is very small compared to vector?
A: For large size differences, you may need to use higher molar ratios to compensate for the mass difference.

Q4: Should I adjust for vector linearization?
A: Yes, always use linearized vector for these calculations. Circular vector calculations would differ.

Q5: How accurate is this calculation?
A: The calculation provides a theoretical estimate. Actual optimal ratios may require empirical testing.