1. What is T4 Ligation?

T4 DNA ligase is an enzyme that catalyzes the formation of phosphodiester bonds between adjacent 5' phosphate and 3' hydroxyl termini in DNA. This calculator helps determine the appropriate mass of insert DNA needed for ligation with your vector.

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 (insert:vector)

Explanation: The equation calculates the mass of insert DNA needed to achieve a specific molar ratio with your vector DNA.

3. Importance of Molar Ratio

Details: Using the correct insert:vector ratio is crucial for successful 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 ratio is set to 3:1.

5. Frequently Asked Questions (FAQ)

Q1: What is the ideal molar ratio for ligation?
A: While 3:1 is commonly used, optimal ratio may vary. For blunt-end ligations, higher ratios (5:1 to 10:1) are often needed.

Q2: How do I measure DNA concentration?
A: Use a spectrophotometer (Nanodrop) or fluorometer (Qubit) for accurate DNA quantification.

Q3: What if my vector and insert have very different sizes?
A: The calculator automatically accounts for size differences through the length parameters.

Q4: Should I use mass or molar ratios?
A: Molar ratios are more important than mass ratios for ligation efficiency.

Q5: What other factors affect ligation efficiency?
A: DNA ends compatibility, ligase concentration, incubation time/temperature, and vector dephosphorylation all affect results.