1. What is the T4 Ligation Formula?

The T4 Ligation formula calculates the required mass of DNA insert (MI) needed for a ligation reaction based on the mass of vector (MV), their respective lengths (LI and LV), and the desired molar ratio (R). This ensures optimal conditions for successful DNA ligation.

2. How Does the Calculator Work?

The calculator uses the T4 Ligation formula:

Where:

• \( MI \) — Insert mass (ng)
• \( MV \) — Vector mass (ng)
• \( LI \) — Insert length (bp)
• \( LV \) — Vector length (bp)
• \( R \) — Molar ratio (typically 3:1 insert:vector)

Explanation: The formula accounts for the molecular weight differences between insert and vector DNA to calculate equivalent molar amounts.

3. Importance of Proper Insert:Vector Ratio

Details: Using the correct insert:vector ratio is crucial for efficient ligation. A 3:1 molar ratio is commonly used to favor insert-vector ligation over vector-vector self-ligation.

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. The default ratio is 3:1 (insert:vector).

5. Frequently Asked Questions (FAQ)

Q1: Why is a 3:1 ratio typically used?
A: A 3:1 insert:vector ratio increases the probability of insert-vector ligation while minimizing vector self-ligation.

Q2: What if my insert is very small compared to the vector?
A: The formula automatically accounts for size differences. Smaller inserts will require less mass to achieve the same molar ratio.

Q3: Can I use different ratios?
A: Yes, you can adjust the ratio. Some protocols use 1:1 to 5:1 depending on the application.

Q4: How accurate does the mass need to be?
A: While precise measurements are ideal, small variations (±10%) are usually acceptable in practice.

Q5: Does this work for blunt-end ligations?
A: Yes, though blunt-end ligations are generally less efficient and might require higher insert:vector ratios.