Promega Ligation Calculator

Ligation Formula:

\[ insert_{mass} = vector_{mass} \times \left(\frac{insert_{length}}{vector_{length}}\right) \times ratio \]

Vector Mass:

Insert Length:

Vector Length:

Insert:Vector Ratio:

unitless

Insert Mass:

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1. What is the Promega Ligation Formula?

The Promega Ligation Formula calculates the optimal mass of DNA insert needed for a ligation 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:

\[ insert_{mass} = vector_{mass} \times \left(\frac{insert_{length}}{vector_{length}}\right) \times ratio \]

Where:

\( insert_{mass} \) — Mass of insert DNA needed (ng)
\( vector_{mass} \) — Mass of vector DNA (ng)
\( insert_{length} \) — Length of insert DNA (bp)
\( vector_{length} \) — Length of vector DNA (bp)
\( ratio \) — Desired molar ratio of insert to vector (unitless)

Explanation: The formula accounts for the relative sizes of the insert and vector to maintain the correct molar ratio in the ligation reaction.

3. Importance of Proper Insert:Vector Ratio

Details: Using the correct ratio of insert to vector is crucial for efficient ligation. A typical ratio of 3:1 (insert:vector) is often used, but optimal ratios may vary depending on the specific application.

4. Using the Calculator

Tips: Enter the vector mass in ng, insert and vector lengths in bp, and the desired molar ratio. All values must be positive numbers.

5. Frequently Asked Questions (FAQ)

Q1: What is the typical insert:vector ratio?
A: A 3:1 ratio is commonly used, but some protocols may recommend ratios from 1:1 to 5:1 depending on the application.

Q2: How do I measure DNA concentrations?
A: Use a spectrophotometer (Nanodrop) or fluorometer (Qubit) to accurately measure DNA concentrations before ligation.

Q3: What if my insert is much smaller than my vector?
A: The calculator automatically accounts for size differences. Smaller inserts will require less mass to achieve the same molar ratio.

Q4: Can I use this for blunt-end ligations?
A: Yes, but blunt-end ligations typically require higher insert:vector ratios (5:1 to 10:1) due to lower efficiency.

Q5: What about sticky-end ligations?
A: The standard 3:1 ratio usually works well for sticky-end ligations, as they are more efficient than blunt-end ligations.