Calculate reagent volumes for PCR, qPCR, and RT-PCR reactions. Add dead volume for pipetting losses.
A master mix combines all common reagents into a single tube before aliquoting into individual reactions. This reduces pipetting steps, minimizes variation between wells, and saves time when setting up large experiments. The master mix typically includes water, buffer, dNTPs, magnesium, and polymerase. Template DNA and primers are added individually per reaction.
Always include dead volume (1-2 extra reactions) to account for pipetting losses. For 96-well plates, add 5-10% overage. Keep the master mix on ice during preparation and aliquot quickly. Mix by gentle vortexing or flicking — never vortex enzymes vigorously.
For qPCR, reaction volumes are typically smaller (10-20 uL) and SYBR Green or probe-based detection replaces ethidium bromide visualization. ROX passive reference dye may be needed depending on the instrument (required for ABI/QuantStudio, not for Bio-Rad CFX).
For strip tubes (8-12 reactions), add 1-2 extra. For 96-well plates, add 5-10% (5-10 extra reactions). For multichannel pipetting from a reservoir, add 15-20% to account for dead volume in the trough. This calculator lets you specify dead volume as extra reaction equivalents.
If all reactions use the same primer pair (e.g., qPCR biological replicates), add primers to the master mix to reduce variation. If different reactions use different primers (e.g., screening multiple targets), add primers individually per well. This calculator marks primers as "per reaction" by default but you can toggle them into the master mix.
Hot-start polymerases are inactive at room temperature and only activate during the initial denaturation step (95C). This prevents nonspecific priming and primer-dimer formation during reaction setup. Use hot-start enzymes for: qPCR, multiplex PCR, low-copy targets, and when setting up many reactions at room temperature.