PCR master mix calculator
Master mix volumes for a batch of PCR reactions, with overage.
Master mix volumes for a batch of PCR reactions, with overage.
Everything that is the same in every tube goes into one mix, which is then split between the tubes; only the template is pipetted separately. Each component is diluted from its stock to its final concentration in the reaction, so its volume follows the dilution rule:
Water makes up whatever is left of the reaction volume once the other components and the template are accounted for. The extra percentage covers what stays behind in the tip and on the walls of the tube; without it the last reaction is always short.
Taq, 25 µL reactions, 12 reactions, 10 % extra, 1 µL of template, which is the built-in example. Each reaction takes 2.50 µL of 10× Standard Taq Buffer, 0.50 µL of 10 mM dNTPs, 0.50 µL of each 10 µM primer, 0.13 µL of Taq at 5 U/µL and 19.88 µL of water, which is 24.00 µL of mix plus 1 µL of template. The mix is made for 12 × 1.10 = 13.2 reactions, so the buffer becomes 33.00 µL, the dNTPs 6.60 µL, each primer 6.60 µL, the Taq 1.65 µL and the water 262.35 µL: 316.80 µL of master mix. Aliquot 24.00 µL per tube and add the template.
| Preset | Buffer | Primers | Enzyme |
|---|---|---|---|
| Taq | 10× Standard Taq | 0.2 µM | 0.025 U/µL |
| OneTaq | 5× OneTaq Standard | 0.2 µM | 0.025 U/µL |
| Phusion | 5× Phusion HF | 0.5 µM | 0.02 U/µL |
| Q5 | 5× Q5 Reaction | 0.5 µM | 0.02 U/µL |
| Pfu | 10× Pfu with MgSO₄ | 0.2 µM | 0.05 U/µL |
| 2× master mix | 2× ready mix | 0.2 µM | in the mix |
All of them assume 10 mM dNTPs and 10 µM primer stocks and give 200 µM dNTPs in the reaction. The buffers carry their own magnesium, so no separate MgCl₂ row appears; if your Taq buffer does not, add 1.5 mM Mg²⁺ from a 25 mM stock, which is 1.5 µL in a 25 µL reaction.
Ten percent is the usual allowance for eight to twenty-four reactions, and it is what this calculator starts with. For fewer than eight reactions use fifteen or twenty percent, because the fixed loss in the tip and the tube is a larger share of a small mix. For a whole 96-well plate five percent is normally enough. Count the no-template control and any positive control as reactions of their own.
Because each reaction gets a different template, and because a common mix contaminated with template ruins every tube including the no-template control. The mix holds buffer, dNTPs, both primers, the polymerase and water; the template goes into each tube separately, which is why the calculator asks how much of it you add and subtracts that volume.
The polymerase supplier decides. NEB specifies 0.2 microMolar for Taq and OneTaq and 0.5 microMolar for Phusion and Q5, and those are the values used here. Raising the primer concentration rarely helps a weak reaction and does make primer dimer more likely, so change the annealing temperature or the template amount first.
Yes, pick the 2x master mix preset. It contains the buffer, dNTPs, magnesium and the polymerase, so the only things you add are primers, template and water. Half the reaction volume is ready mix; the calculator works out the rest.
Only for templates with strong secondary structure or high GC content. Three percent final is a common starting point with Phusion and Q5, and it lowers the effective annealing temperature by roughly a degree for each percent, so drop the annealing temperature when you add it. Enter the final percentage under the result and it appears in the table as neat DMSO.