PCR Master Mix Calculator

PCR (Polymerase Chain Reaction) is a lab technique used to amplify DNA, and preparing a master mix — a single batch of all shared reagents — saves time and reduces pipetting errors across multiple reactions. Enter your single reaction volume, number of reactions, and overage value, then set the stock and final concentrations for each component — PCR buffer, dNTPs, forward and reverse primers, Taq polymerase, and optionally MgCl₂. The PCR Master Mix Calculator returns the total master mix volume plus the exact µL amount of each reagent and water to add. Also try the GC Content Calculator.

µL

Final volume for one individual PCR tube

Number of samples/tubes excluding controls

Extra volume for pipetting safety margin

µL

Volume of template added separately to each tube

x
x
mM
mM
µM
µM
µM
µM
U/µL
U/µL
mM
mM

Results

Total Master Mix Volume

--

PCR Buffer Volume

--

dNTP Volume

--

Forward Primer Volume

--

Reverse Primer Volume

--

Taq Polymerase Volume

--

MgCl₂ Volume

--

Water Volume

--

Master Mix per Reaction

--

Results Table

Frequently Asked Questions

What is overage and why should I include it in my PCR master mix calculations?

Overage is extra volume added to compensate for pipetting losses and ensure you have enough master mix for all reactions. It's typically 5-15% of the total volume or 1-2 extra reactions worth of reagents. See also our DNA/RNA Molecular Weight Calculator.

How do I determine the optimal primer concentrations for my PCR?

Standard primer concentrations range from 0.1-1.0 µM final concentration. Start with 0.5 µM and optimize based on your specific primers and template. Higher concentrations may increase non-specific amplification.

Should I include MgCl₂ separately if my buffer already contains it?

Most modern PCR buffers include MgCl₂ at optimal concentrations. Only add separate MgCl₂ if using a buffer without it or if optimization requires different Mg²⁺ concentrations (typically 1-4 mM final).

What's the difference between stock and final concentrations?

Stock concentration is the concentration of your reagent as purchased or prepared. Final concentration is the desired concentration in your PCR reaction after dilution with other components.

How much Taq polymerase should I use per reaction?

Typical Taq polymerase usage is 0.025-0.05 U/µL final concentration (1.25-2.5 U per 50 µL reaction). Using too much can increase non-specific amplification and cost.

Can I prepare master mix in advance and store it?

Fresh master mix is always best, but it can be stored at -20°C for up to one week or 4°C for 24 hours. Add template DNA just before use and mix gently to avoid introducing bubbles.

What should I do if I don't have enough volume for accurate pipetting?

If calculated volumes are too small for accurate pipetting (< 0.5 µL), consider scaling up your reaction volume, using more concentrated stocks, or preparing a larger batch and aliquoting.

How do I account for dead volume in tubes and tips?

The overage setting helps account for dead volumes. For very small volumes, consider the ~0.5-1 µL that typically remains in pipette tips and tube bottoms when calculating your overage needs.