Ligation Calculator Neb

Ligation Formula:

\[ \text{insert\_mass} = \text{vector\_mass} \times \left(\frac{\text{insert\_length}}{\text{vector\_length}}\right) \times \text{ratio} \]

Vector Mass:

Insert Length:

Vector Length:

Insert:Vector Ratio:

(unitless)

Required Insert Mass:

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1. What is the Ligation Calculation?

The ligation calculation determines the optimal mass of DNA insert needed for a cloning reaction based on the mass of vector DNA, their respective lengths, and the desired molar ratio of insert to vector.

2. How Does the Calculator Work?

The calculator uses the ligation formula:

\[ \text{insert\_mass} = \text{vector\_mass} \times \left(\frac{\text{insert\_length}}{\text{vector\_length}}\right) \times \text{ratio} \]

Where:

\( \text{insert\_mass} \) — Required mass of insert DNA (ng)
\( \text{vector\_mass} \) — Mass of vector DNA (ng)
\( \text{insert\_length} \) — Length of insert DNA (bp)
\( \text{vector\_length} \) — Length of vector DNA (bp)
\( \text{ratio} \) — Desired molar ratio of insert to vector (typically 3:1)

Explanation: The equation accounts for the different molecular weights of the insert and vector based on their lengths to calculate the mass needed to achieve the desired molar ratio.

3. Importance of Proper Ligation Ratios

Details: Using the correct insert:vector ratio is crucial for efficient ligation. Too little insert results in mostly empty vector, while too much can lead to multiple inserts or other unwanted products.

4. Using the Calculator

Tips: Enter vector mass in ng, lengths in bp, and desired ratio (typically 3:1). All values must be positive numbers. The default ratio of 3:1 is recommended by NEB for most applications.

5. Frequently Asked Questions (FAQ)

Q1: What is the typical insert:vector ratio?
A: A 3:1 molar ratio of insert to vector is commonly recommended, though some protocols may suggest 1:1 to 10:1 depending on the application.

Q2: How accurate does the DNA quantification need to be?
A: For best results, use fluorometric methods (like Qubit) rather than spectrophotometry (Nanodrop) for more accurate DNA quantification.

Q3: Should I adjust for different DNA forms?
A: This calculation assumes linear DNA. For circular or supercoiled DNA, additional adjustments may be needed.

Q4: What if my vector and insert have very different sizes?
A: The calculation automatically accounts for size differences through the length ratio term.

Q5: Can I use this for Gibson assembly?
A: While similar principles apply, Gibson assembly typically uses equimolar ratios (1:1) of all fragments including the vector.