1. What is Plasmid Ligation?

Plasmid ligation is the process of joining DNA fragments (inserts) into plasmid vectors using DNA ligase. The correct ratio of insert to vector is crucial for efficient cloning.

2. How Does the Calculator Work?

The calculator uses the plasmid ligation formula:

Where:

• \( Insert_{ng} \) — Mass of insert DNA needed (ng)
• \( vector_{ng} \) — Mass of vector DNA (ng)
• \( kb_{insert} \) — Size of insert DNA (kb)
• \( kb_{vector} \) — Size of vector DNA (kb)
• \( molar_{ratio} \) — Desired insert:vector molar ratio

Explanation: The equation calculates the mass of insert DNA needed to achieve a specific molar ratio with your vector DNA, accounting for their size differences.

3. Importance of Molar Ratios

Details: Using the correct insert:vector ratio (typically 3:1 to 5:1) prevents self-ligation of vectors and increases cloning efficiency. Too much insert can lead to multiple inserts, while too little reduces positive clones.

4. Using the Calculator

Tips: Enter vector mass in ng, insert and vector sizes in kb, and desired molar ratio. Typical ratios are 3:1 for standard cloning or 1:1 for blunt-end ligations.

5. Frequently Asked Questions (FAQ)

Q1: What's a typical molar ratio for ligation?
A: 3:1 insert:vector is standard for sticky-end ligations. Blunt-end ligations may require higher ratios (5:1 to 10:1).

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

Q3: What if my insert is very small/large?
A: For very small inserts (<200bp), you may need higher ratios. For large inserts (>5kb), lower ratios may work better.

Q4: Should I adjust for vector dephosphorylation?
A: Yes, if using dephosphorylated vector, you may need to increase the insert amount by 2-3 fold.

Q5: What's the total reaction volume?
A: This calculator gives DNA masses. Typical ligation reactions are 10-20μL total volume with 50-100ng total DNA.