1. What is Plasmid Copy Number?

The plasmid copy number (CN) represents the number of copies of a plasmid per host cell. This calculator estimates the relative copy number from quantitative PCR (qPCR) cycle threshold (Ct) values using a reference Ct value.

2. How Does the Calculator Work?

The calculator uses the equation:

Where:

• \( CN \) — Copy number (dimensionless)
• \( Ct_{ref} \) — Reference cycle threshold (dimensionless)
• \( Ct \) — Plasmid cycle threshold (dimensionless)

Explanation: The equation assumes perfect PCR efficiency (100%) where each cycle doubles the DNA amount. The difference in Ct values between reference and sample is used to calculate relative copy number.

3. Importance of Copy Number Calculation

Details: Knowing plasmid copy number is essential for genetic engineering, protein expression studies, and ensuring consistent experimental results in molecular biology.

4. Using the Calculator

Tips: Enter both Ct values (reference and plasmid) from your qPCR experiment. The values should be from the same run under identical conditions.

5. Frequently Asked Questions (FAQ)

Q1: What is a good reference Ct value?
A: Typically use a known single-copy plasmid or genomic DNA as reference. The choice depends on your experimental design.

Q2: What if my PCR efficiency isn't 100%?
A: For non-ideal efficiency, use the equation: CN = (1 + E)^{(Ct_ref - Ct)}, where E is the efficiency (0 to 1).

Q3: What are typical plasmid copy numbers?
A: Low-copy plasmids (1-10 copies/cell), medium-copy (10-20), and high-copy (20-100+) are common, depending on origin of replication.

Q4: Can I use this for genomic DNA?
A: This method is designed for plasmids. For genomic DNA, different normalization methods are typically used.

Q5: How precise is this calculation?
A: This provides a relative estimate. For absolute quantification, standard curves with known copy numbers are recommended.