1. What is the NEB Variant 2 Equation?

The NEB Variant 2 equation calculates the required insert mass (MI) for a ligation reaction based on the vector mass (MV), insert length (LI), vector length (LV), and desired molar ratio (R). This is commonly used in molecular biology for DNA ligation experiments.

2. How Does the Calculator Work?

The calculator uses the NEB Variant 2 equation:

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 accounts for the relative masses and lengths of the insert and vector DNA to achieve the desired molar ratio in the ligation reaction.

3. Importance of Molar Ratio in Ligation

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 3:1.

5. Frequently Asked Questions (FAQ)

Q1: What is a typical molar ratio for ligation?
A: Most standard ligations use a 3:1 insert:vector molar ratio, though this may vary depending on the application.

Q2: How do I measure vector and insert lengths?
A: Lengths should be in base pairs (bp) and can be determined from gel electrophoresis or known sequence information.

Q3: What if I have multiple inserts?
A: For multiple inserts, calculate each separately and combine them in the desired ratios.

Q4: Does this work for blunt-end ligations?
A: Yes, though blunt-end ligations often require higher molar ratios (5:1 to 10:1) for efficient cloning.

Q5: How accurate is this calculation?
A: The calculation provides a theoretical estimate. Actual results may vary based on DNA quality, ends compatibility, and ligation efficiency.