Promega Ligation Calculator Malaysia

Promega Ligation Formula:

\[ MI = \frac{MV \times LI}{LV} \times R \]

Vector Mass (MV):

Insert Length (LI):

Vector Length (LV):

Molar Ratio (R):

Insert Mass (MI):

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1. What is the Promega Ligation Calculator?

The Promega Ligation Calculator helps determine the appropriate mass of DNA insert needed for a ligation reaction based on the vector mass, lengths of both fragments, and desired molar ratio. This is particularly useful for molecular cloning experiments.

2. How Does the Calculator Work?

The calculator uses the Promega ligation formula:

\[ MI = \frac{MV \times LI}{LV} \times R \]

Where:

\( MI \) — Insert mass (ng)
\( MV \) — Vector mass (ng)
\( LI \) — Insert length (bp)
\( LV \) — Vector length (bp)
\( R \) — Molar ratio (typically 3:1 insert:vector)

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

3. Importance of Proper Ligation Ratios

Details: Using the correct insert:vector ratio is crucial for successful ligation. A 3:1 molar ratio is commonly used to favor insert-vector ligation over vector-vector self-ligation.

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 of 3:1 is recommended for most applications.

5. Frequently Asked Questions (FAQ)

Q1: What is the typical molar ratio for ligation?
A: A 3:1 insert:vector ratio is standard, though some protocols may recommend 2:1 to 5:1 depending on the application.

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 vector and insert have very different lengths?
A: The calculator automatically accounts for length differences through the formula. Longer fragments require more mass to achieve the same molar ratio.

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

Q5: What about PCR product ligations?
A: PCR products should be purified before ligation. Consider adding 15-30% extra insert to account for potential inaccuracies in quantification.