How to use (3 steps)
- Choose an example or enter reaction volume and reaction count (or 96/384 well plate).
- Set overage (e.g., 10%) and components (2× mix, primers, etc.).
- Per-reaction volumes, totals with overage, and a pipetting protocol are shown.
This tool calculates volumes only. Optimize conditions separately.
Inputs
Results
Static sample: 10 reactions, 25 µL each, 10% overage, 2× master mix, primers, template added separately. JavaScript recalculates the same table from your inputs.
Component table
| Component | Per reaction (µL) | Total (µL) | Notes |
|---|---|---|---|
| 2× Master Mix | 12.5 | 137.5 | |
| Forward primer | 0.5 | 5.5 | |
| Reverse primer | 0.5 | 5.5 | |
| Template | 1 | 10 | Separate |
| Water | 10.5 | 115.5 | Fill |
Table scrolls horizontally.
“Separate” indicates components typically added outside the master mix (e.g., template).
Protocol (copy-ready)
PCR master mix protocol (CalcBE) Prepare: 11 reactions (actual wells=10) 1) Prepare the master mix - 2× Master Mix: 137.5 µL - Forward primer: 5.5 µL - Reverse primer: 5.5 µL - Water: 115.5 µL 2) Dispense 24 µL of master mix into each well 3) Add template 1 µL per well This tool calculates volumes only. Optimize conditions separately.
Share URL / Export
Share URLs include inputs, overage, template mode, and component settings. They can be long, but the calculation still runs in your browser.
Share URLs include inputs, overage, template mode, and component settings; no calculation data is sent to a server.
How it's calculated
- From concentration to volume: Using C1V1=C2V2, V1 = (C_final / C_stock) × Vtotal.
- Fold concentration (2×/5×): V = (final_x / stock_x) × Vtotal (usually final = 1×).
- Overage: Percentage is rounded up (ceil), prepared = ceil(wells × (1 + pct/100)).
- Water (fill): Water = Vtotal − ΣV(component). Negative means an error.
- Totals: Components included in the master mix use prepared count; separate components use wells count (toggleable).
This tool calculates volumes only; it does not guarantee experimental success.
FAQ
What is a master mix?
A master mix is a common reagent mix prepared for multiple reactions. It reduces pipetting steps and variability.
How much overage should I add?
A typical guide is 5–10% or +1 reaction. Adjust based on pipetting and reaction count.
How do I enter a 2× (or 5×) master mix?
Enter it as a fold concentration. For a 2× mix to reach 1× final, the volume is about half of the reaction volume (computed automatically).
Should the template be included in the master mix?
Often the template is added separately to reduce contamination risk and sample-to-sample bias. This tool defaults to separate addition (toggleable).
Why is the water volume negative?
The component totals exceed the reaction volume. Increase reaction volume or reduce component volumes (especially template).
I got a very small volume (e.g., 0.1 µL).
Such volumes are hard to pipette. Consider intermediate dilution or adjusting the reaction volume.
Can I use this for qPCR?
Yes. Enter a 2× qPCR mix and primer concentrations to compute total volumes.
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