1. What is the NEB T4 Ligase Calculator?

The NEB T4 Ligase Calculator helps determine the appropriate mass of insert DNA needed for a ligation reaction based on the vector mass, lengths of insert and vector, and desired molar ratio.

2. How Does the Calculator Work?

The calculator uses the following 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 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 insert:vector molar ratio 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 default molar ratio?
A: The calculator defaults to 3:1 (insert:vector), which works well for most applications.

Q2: How do I determine vector mass?
A: Measure vector concentration (ng/μL) by spectrophotometry and multiply by the volume you plan to use.

Q3: What if I have multiple inserts?
A: Calculate each insert separately using the same vector mass and length.

Q4: Does this work for blunt-end ligations?
A: Yes, but blunt-end ligations typically require higher insert:vector ratios (5:1 to 10:1).

Q5: What about T4 DNA Ligase buffer?
A: This calculator only determines insert mass. Always follow manufacturer's instructions for buffer composition.