1. What is In-Fusion Cloning?

In-Fusion cloning is a seamless cloning technique that allows precise joining of DNA fragments without restriction enzymes or ligases. It relies on homologous recombination between 15-20 bp overlapping ends of the insert and vector.

2. How Does the Calculator Work?

The calculator uses the molar ratio formula:

Where:

• insert — Amount of insert DNA needed (ng)
• vector — Amount of vector DNA used (ng)
• ratio — Desired molar ratio (typically 2:1 for insert:vector)
• insert_length — Length of insert DNA (bp)
• vector_length — Length of vector DNA (bp)

Explanation: The calculation ensures equimolar amounts of DNA fragments by accounting for their different molecular weights based on length.

3. Importance of Molar Ratio

Details: Using the correct molar ratio is crucial for efficient cloning. A 2:1 insert:vector ratio is typically recommended to favor insert-vector recombination over vector self-ligation.

4. Using the Calculator

Tips: Enter vector amount in ng, desired molar ratio (typically 2), and the lengths of both insert and vector in base pairs. All values must be positive numbers.

5. Frequently Asked Questions (FAQ)

Q1: What molar ratio should I use?
A: For standard In-Fusion cloning, a 2:1 insert:vector ratio is recommended. For difficult clones, you may try 3:1 or higher.

Q2: How do I measure DNA concentrations?
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 inserts <100 bp or >5 kb, you may need to adjust the ratio. Very small inserts may require higher ratios (3:1 to 5:1).

Q4: Can I use this for Gibson Assembly?
A: Yes, the same molar ratio principles apply to Gibson Assembly and other seamless cloning methods.

Q5: What's the minimum vector amount I should use?
A: Typically use at least 50 ng of vector for good transformation efficiency, adjusting insert amount accordingly.