1. What is T4 DNA Ligase?

T4 DNA Ligase is an enzyme that catalyzes the formation of phosphodiester bonds between adjacent 5' phosphate and 3' hydroxyl termini in DNA. It's commonly used in molecular biology for DNA cloning and ligation reactions.

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 the vector DNA in the ligation reaction.

3. Importance of Molar Ratios

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

5. Frequently Asked Questions (FAQ)

Q1: What is the optimal molar ratio for ligation?
A: While 3:1 is commonly used, the optimal ratio may vary depending on insert and vector characteristics. Some protocols recommend testing ratios from 1:1 to 10:1.

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

Q3: What if my insert is very small or very large?
A: For very small inserts (<100bp), you may need higher ratios. For very large inserts, consider lower ratios to avoid excessive DNA mass.

Q4: Should I adjust for vector dephosphorylation?
A: Yes, if using dephosphorylated vector, you may need to adjust ratios as this prevents vector self-ligation.

Q5: What about sticky vs blunt ends?
A: Blunt end ligations typically require more DNA and higher ratios than sticky end ligations.