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 In-Fusion cloning 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 the desired molar ratio with your vector DNA.

3. Importance of Molar Ratios

Details: Using the correct molar ratio is crucial for efficient cloning. Typical ratios range from 1:1 to 5:1 (insert:vector). Too much insert can lead to multiple inserts, while too little reduces cloning efficiency.

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 1:1.

5. Frequently Asked Questions (FAQ)

Q1: What's a typical molar ratio for In-Fusion cloning?
A: Most protocols recommend a 2:1 insert:vector molar ratio for optimal results.

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

Q3: What if my insert is very small or very large?
A: For very small inserts (<100 bp), you may need to increase the molar ratio. For very large inserts, consider using more DNA.

Q4: Can I use this for Gibson Assembly?
A: Yes, the same calculation applies to Gibson Assembly and other seamless cloning methods.

Q5: What about PCR product purification?
A: Always purify PCR products before cloning to remove primers and polymerase that could interfere with the reaction.