1. What is the NEB Ligation Calculator?

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

2. How Does the Calculator Work?

The calculator uses the NEB 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 in Ligation

Details: Using the correct molar ratio of insert to vector is crucial for efficient ligation. A typical ratio ranges from 1:1 to 10:1 (insert:vector), with 3:1 being commonly used.

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 the recommended molar ratio for ligation?
A: For most applications, a 3:1 insert:vector molar ratio works well. You may need to optimize this for specific cases.

Q2: How do I measure vector mass?
A: Use a spectrophotometer (Nanodrop) or fluorometer (Qubit) to quantify your vector DNA concentration.

Q3: What if my insert is much smaller than my vector?
A: The calculator automatically accounts for size differences through the LI/LV ratio term.

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

Q5: How accurate does the ratio need to be?
A: While the calculation is precise, the biological system has some flexibility. Being within 20% of the target ratio is usually sufficient.