13.2
23
µL
303.6
µL
13.2
23
µL
303.6
µL
The PCR Master Mix Calculator determines the total volume of master mix to prepare for multiple PCR reactions, including an overage for pipetting losses. Master mix preparation is a routine but error-prone step in PCR workflows. By pooling common reagents into a single mix, you reduce pipetting steps, improve consistency between reactions, and minimize contamination risk. This calculator accounts for template volume that is added separately to each tube.
The calculator computes:
Effective Reactions = Number of Reactions × (1 + Extra %/100)
Master Mix per Reaction = Total Reaction Volume – Template Volume
Total Master Mix = Master Mix per Reaction × Effective Reactions
The extra percentage (typically 10%) accounts for volume lost to pipette tips and tube walls. The template DNA is added separately to each reaction tube after aliquoting the master mix.
Inputs
Results
For 12 reactions at 25 µL each (2 µL template), prepare 303.6 µL of master mix (13.2 effective reactions with 10% extra).
Inputs
Results
A full 96-well plate at 20 µL reactions requires approximately 2006.4 µL (about 2 mL) of master mix.
Each time you pipette, a small amount of liquid remains on the tip and tube walls. Over many aliquots, this loss accumulates and the last few reactions may not receive enough master mix. Adding 10% extra (or 1–2 extra reactions) ensures you have sufficient volume. For viscous reagents or many reactions, increase to 15–20% extra.
A standard PCR master mix contains: PCR buffer (1× final), dNTPs (200 µM each), forward and reverse primers (200–500 nM each), DNA polymerase (0.5–2.5 U per reaction), MgCl2 (1.5–3 mM), and nuclease-free water to the final volume. Template DNA is added separately.
No. Template DNA should always be added separately to each reaction tube after dispensing the master mix. This ensures each reaction receives the correct template and prevents cross-contamination. It also allows you to include no-template controls (NTC) from the same master mix batch.
Roboculator Team
The Roboculator Team explains calculations, planning tools, and practical formulas in clear language for real-life situations.
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