1. What is Primer Concentration?

Primer concentration refers to the molarity (μM) of primer molecules in a solution. It's a critical parameter in molecular biology experiments, especially in PCR applications where optimal primer concentration is 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 give concentration in μM.

3. Importance of Primer Concentration

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

4. Using the Calculator

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

5. Frequently Asked Questions (FAQ)

Q1: What is a typical primer concentration for PCR?
A: Most PCR reactions use primer concentrations between 0.1-1 μM, with 0.5 μM being a common starting point.

Q2: How do I convert OD260 to nmol for primers?
A: nmol = (OD260 × dilution factor × 1000) / (extinction coefficient). The manufacturer typically provides this information.

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

Q4: 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.

Q5: What if my calculated concentration seems too high or low?
A: Double-check your inputs. Typical primer synthesis yields range from 10-50 nmol, and resuspension volumes are usually 100-500 μL.