1. What is Primer Concentration?

Primer concentration refers to the molarity (μM) of oligonucleotide primers in solution. It's a critical parameter in molecular biology experiments like PCR, where optimal primer concentrations are essential for successful amplification.

2. How Does the Calculator Work?

The calculator uses the primer concentration formula:

Where:

• \( C \) — Primer concentration (μM)
• \( nmol \) — Amount of primer (nmol)
• \( V \) — Volume of solution (μL)

Explanation: The formula converts the amount of primer from nmol to pmol (by multiplying by 1000) and divides by the volume in μL to get concentration in μM (μmol/L).

3. Importance of Primer Concentration

Details: Proper primer concentration is crucial for PCR efficiency. Too low concentration may result in no amplification, while too high concentration can lead to non-specific binding and primer-dimer formation.

4. Using the Calculator

Tips: Enter the amount of primer in nmol (as typically provided by synthesis companies) and the volume of solution in μL. Both values must be positive numbers.

5. Frequently Asked Questions (FAQ)

Q1: What is a typical working concentration for PCR primers?
A: Most PCR reactions use primers at 0.1-1 μM final concentration, with 0.5 μM being a common starting point.

Q2: How do I convert OD units to nmol for primer calculations?
A: Use the formula: nmol = OD × 33 × µg/nmol, where 33 is the average extinction coefficient for DNA.

Q3: Should I use the same concentration for both forward and reverse primers?
A: Generally yes, unless specifically optimizing asymmetric PCR. Equal concentrations (within 10%) are recommended.

Q4: How does primer concentration affect PCR efficiency?
A: Optimal concentration ensures specific binding. Too high can cause non-specific products, too low may reduce yield.

Q5: How should I store primer stock solutions?
A: Store at -20°C in TE buffer or nuclease-free water. Avoid repeated freeze-thaw cycles by making aliquots.