1. What is Plasmid Copy Number?

The plasmid copy number (CN) represents the number of copies of a plasmid present per bacterial cell. It's an important parameter in molecular biology that affects gene expression levels and plasmid stability.

2. How Does the Calculator Work?

The calculator uses the relative quantification equation:

Where:

• \( CN \) — Copy number (dimensionless)
• \( E \) — PCR efficiency (1-2)
• \( Ct \) — Threshold cycle of sample
• \( Ct_{ref} \) — Threshold cycle of reference

Explanation: The equation calculates the relative copy number based on the difference in Ct values between the sample and reference, adjusted for PCR efficiency.

3. Importance of Copy Number Calculation

Details: Knowing plasmid copy number is crucial for experimental design, ensuring consistent gene expression, and maintaining plasmid stability in bacterial cultures.

4. Using the Calculator

Tips: Enter PCR efficiency (typically 1.8-2.0 for optimal reactions), sample Ct value, and reference Ct value. All values must be valid (efficiency between 1-2).

5. Frequently Asked Questions (FAQ)

Q1: What is a typical plasmid copy number?
A: Copy numbers vary widely from 1-2 (low copy) to 50+ (high copy) plasmids per cell, depending on the origin of replication.

Q2: How accurate is this calculation method?
A: The method provides a relative estimate. For absolute quantification, standard curves with known copy numbers are recommended.

Q3: What PCR efficiency should I use?
A: Ideally determine your own PCR efficiency experimentally. For perfect doubling, use 2.0.

Q4: Can I use this for genomic DNA?
A: This method is designed for plasmid copy number. Different calculations are needed for genomic DNA.

Q5: What reference should I use?
A: Typically use a single-copy chromosomal gene as reference for plasmid copy number determination.