1. What is the NEB Molar Ratio?

The NEB (New England Biolabs) molar ratio calculates the optimal insert:vector ratio for ligation reactions. It accounts for both the mass and length of DNA fragments to ensure proper stoichiometry.

2. How Does the Calculator Work?

The calculator uses the NEB molar ratio equation:

Where:

• \( ng\ insert \) — Mass of insert DNA in nanograms
• \( kb\ insert \) — Length of insert DNA in kilobases
• \( ng\ vector \) — Mass of vector DNA in nanograms
• \( kb\ vector \) — Length of vector DNA in kilobases

Explanation: The equation normalizes DNA amounts by length before calculating their ratio, ensuring equal molar representation of fragments.

3. Importance of Molar Ratio in Ligation

Details: Proper insert:vector ratio is crucial for efficient ligation. Typical ratios range from 1:1 to 3:1 (insert:vector). Too much insert can lead to multiple inserts, while too little reduces cloning efficiency.

4. Using the Calculator

Tips: Enter all values in the specified units (ng for mass, kb for length). For best results, use accurate measurements from gel quantification or spectrophotometry.

5. Frequently Asked Questions (FAQ)

Q1: What is the ideal molar ratio for ligation?
A: Most protocols recommend 3:1 insert:vector ratio, but optimal ratio may vary based on fragment ends and application.

Q2: How do I measure DNA concentration and length?
A: Use nanodrop for concentration and gel electrophoresis with size standards for length estimation.

Q3: Should I adjust for different DNA forms?
A: Yes, linearized vector works best. Supercoiled vectors require different calculations.

Q4: What if my ratio is very high?
A: High ratios (>5:1) may increase background. Consider reducing insert amount or increasing vector.

Q5: Does this work for blunt-end ligations?
A: Blunt-end ligations often require higher insert:vector ratios (up to 10:1) due to lower efficiency.