How to Calculate PCR Mastermix Volumes Accurately

Polymerase Chain Reaction (PCR) is a fundamental technique in molecular biology. One of the most common sources of error and waste is incorrect calculation of the Mastermix.

When setting up multiple PCR reactions, it is standard practice to create a Mastermix containing all common reagents (Water, Buffer, dNTPs, Primers, Polymerase) and then aliquot this into individual tubes. This ensures consistency across samples.

The Basic Formula

For a single reaction, you might have:

Water: 15.5 µL
Buffer (10x): 2.5 µL
dNTPs (10mM): 0.5 µL
Forward Primer: 1.0 µL
Reverse Primer: 1.0 µL
Taq Polymerase: 0.5 µL
Total Volume: 21.0 µL (add 4 µL template later)

If you have 10 samples, you might think you just multiply everything by 10. However, this is where most beginners fail.

The "N+1" Rule

Pipetting is never perfect. Liquid sticks to the outside of tips, or slight calibration errors occur. If you prepare exactly enough for 10 samples, the last tube will likely be short.

Always calculate for N + 10% or simply N + 1 samples.

For 10 samples, calculate for 11.
For 96 samples (plate), calculate for 100-105.

Avoid Calculation Errors

Use our free PCR Mastermix Calculator to automatically apply the 10% safety margin.

Open PCR Calculator

Handling Viscous Reagents

Reagents like Glycerol (often in Taq buffer) or pure enzymes are viscous.

Pipette slowly: Wait for the liquid to equilibrate.
Reverse pipette: If highly viscous.
Mix gently: Do not vortex enzymes vigorously; invert the tube or flick gently.

Summary Checklist

[ ] Determine total reaction volume (usually 25 or 50 µL)
[ ] List all reagents and their stock concentrations
[ ] Determine final concentrations required
[ ] Calculate volume per reaction: V1 = (C2 * V2) / C1
[ ] Multiply by (Samples + Error Margin)
[ ] Add water first, enzyme last.

Happy cycling!