1. What is the NEB Ligation Formula?

The NEB (New England Biolabs) Ligation Formula calculates the required mass of DNA insert (MI) for optimal ligation with a vector, based on their lengths and desired molar ratio.

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 formula accounts for the molecular weight differences between insert and vector to achieve the desired molar ratio in the ligation reaction.

3. Importance of Molar Ratios in Ligation

Details: Proper molar ratios are crucial for efficient ligation. Typical ratios range from 1:1 to 10:1 (insert:vector), with 3:1 being most common for standard cloning.

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 3:1 (insert:vector).

5. Frequently Asked Questions (FAQ)

Q1: What's the ideal molar ratio for ligation?
A: For most applications, 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 vector mass accurately?
A: Use nanodrop or Qubit quantification after gel purification or column cleanup for most accurate results.

Q3: Does fragment purity affect ligation efficiency?
A: Yes, contaminants like salts, EDTA, or agarose can inhibit ligation. Always use clean DNA fragments.

Q4: What if my insert is very small compared to vector?
A: For small inserts (<100bp), you may need to increase the ratio (5:1 to 10:1) due to lower ligation efficiency.

Q5: How much total DNA should be in my ligation reaction?
A: Typically 50-100ng of vector DNA per 20μL reaction, but follow your ligase manufacturer's recommendations.