PCR Master Mix Calculator

1. Choose 2× Master Mix or Custom components.
2. Set final reaction volume, # reactions, and overage.
3. Enter your reagent inputs and click Calculate.
4. Use the table to prepare your master mix (usually add template last).

• If your polymerase is a hot-start enzyme, follow kit guidance for setup and cycling.
• If you’re using a 2× Master Mix, many components are already included (buffer, Mg²⁺, dNTPs, enzyme).
• Always keep enzymes cold and spin down tubes before pipetting.

• 2× mode: Sets V(2×) (usually Vfinal/2), then subtracts primers + template and fills the rest with water.
• Custom mode: Uses C₁V₁ = C₂V₂ to compute each reagent volume per reaction, then fills remaining volume with water.
• Totals: Multiplies each per-reaction volume by N × (1 + overage%).

Lab note: The “master mix” total shown excludes template (so you can add template separately, usually last).

Example 1 — 2× Master Mix mode

Make 8 reactions of 25 µL PCR with 10% overage. Use standard 2× mix volume (12.5 µL), primers 0.5 µL each, template 1 µL.

1. Per reaction volumes: 2×=12.5, Fwd=0.5, Rev=0.5, Template=1.0
2. Water per reaction: 25 − (12.5 + 0.5 + 0.5 + 1.0) = 10.5 µL
3. Overage factor: F = 1 + 10/100 = 1.10 → total multiplier 8 × 1.10 = 8.8
4. Total 2× mix: 12.5 × 8.8 = 110.0 µL (similarly for other components)

Example 2 — Custom components mode

Final volume 25 µL. Buffer 10×→1×, MgCl₂ 50 mM→1.5 mM, dNTPs 10 mM each→0.2 mM each, primers 10 µM→0.5 µM each, polymerase 5 U/µL and 0.5 U/reaction, template 1 µL.

1. Buffer: V = (1/10)×25 = 2.5 µL
2. MgCl₂: V = (1.5/50)×25 = 0.75 µL
3. dNTPs: V = (0.2/10)×25 = 0.5 µL
4. Primer each: V = (0.5/10)×25 = 1.25 µL (forward + reverse)
5. Polymerase: V = 0.5/(5) = 0.1 µL
6. Water: 25 − (2.5+0.75+0.5+1.25+1.25+0.1+1.0) = 17.65 µL