PCR Master Mix

Assumptions

PCR master mix calculator for one double-stranded DNA amplification setup. Enter the number of reactions, an overage percent, and the reaction volume. Each component has a stock and a final amount. Water fills whatever volume is left. The page does not choose an annealing temperature, and it does not replace the polymerase manual.

The numbers filled in on first load are a Standard 50 µL Taq PCR: a 50 µL reaction with 1X buffer from a 10X stock, 200 µM each dNTP from a 10 mM mix, 0.2 µM of each primer from a 10 µM stock, and 1.25 units of polymerase. At 5 U/µL that polymerase volume is 0.25 µL. Standard Taq buffer in this recipe already contains magnesium, so MgCl2 is left blank unless you are using a magnesium-free buffer. Follow the sheet that came with your own enzyme if it differs. The citation is under Sources. This page is not a product page.

Overage is an extra percent, not a count of spare reactions. Ten reactions at 10% is eleven reaction-equivalents of master mix. The extra mix covers pipette loss. Template that is added to each tube is not multiplied by that overage: you still add it only to the reactions you set up. Put template in the mix only when you uncheck that option.

How it works

µL = reaction µL × final ÷ stock

polymerase µL = U per reaction ÷ stock U/µL

water = reaction − the other components

Buffer, dNTPs, primers, and optional MgCl2 use the same relation as a single dilution: microlitres equal the reaction volume times the final concentration divided by the stock. dNTPs and primers are converted with the shared molar scale first, so 10 mmol/L and 200 µmol/L meet in mol/L before the division. Polymerase is not a concentration in the reaction volume. Its volume is the units you want in one reaction divided by the stock in units per microlitre.

Water is the reaction volume minus every other per-reaction volume, including template. If that remainder is negative, the components do not fit and the page shows no table. A final amount above its stock is rejected before that check, because one component would already be larger than the tube. A final amount equal to the stock is allowed and uses the whole reaction for that component. Display is four significant figures, the same rounding as the solution channels.

The master-mix total multiplies each in-mix volume by the reaction count and by (100 + overage) / 100. Template kept out of the mix is multiplied only by the reaction count. The LCD is the volume of mix to dispense into one tube: the full reaction when template is in the mix, otherwise the reaction minus the template volume. Copy result is the tab-separated table. Copy protocol is the longer text and still starts with Molicheck PCR master mix protocol.

Components that go into the master mix are compared using their total volume, overage included. Template added to each tube, and the volume dispensed into each tube, are compared one tube at a time. A volume strictly below the minimum is marked, and the result stays on screen. The status line names that component. An equal volume is not below the minimum. Leave the minimum blank and the page does not compare. The comparison uses the shared volume scale, the same strict test as C1V1 and serial dilution.

Worked example

Reactions 10, overage 10%, reaction volume 50 µL. Dispense 49.00 µL. Polymerase is 0.2500 µL per reaction (2.750 µL total). Water is 40.75 µL per reaction (448.3 µL total). Template is 1.000 µL per tube (10.00 µL total) and is not in the master mix.

• mix reactions = 10 × (100 + 10) ÷ 100
• Reaction buffer = 5.000 µL per reaction, 55.00 µL total
• dNTPs = 1.000 µL per reaction, 11.00 µL total
• Forward primer = 1.000 µL per reaction, 11.00 µL total
• Reverse primer = 1.000 µL per reaction, 11.00 µL total
• Polymerase = 0.2500 µL per reaction, 2.750 µL total
• Water = 40.75 µL per reaction, 448.3 µL total
• Template DNA = 1.000 µL per reaction, 10.00 µL total
• dispense = 49.00 µL per tube
• template is added per tube and is not in the master mix

1. Prepare master mix for 10 reactions plus 10% overage.
2. Dispense 49.00 µL of master mix into each tube.
3. Add 1.000 µL of template to each tube.

Common mistakes

Adding magnesium that this standard Taq recipe already includes in the buffer. A second MgCl2 row double-counts it. Use the optional row for a magnesium-free buffer, and then enter the stock and final from that enzyme’s own manual.

Checking the 0.25 µL polymerase volume as if you pipette it once per tube. The master-mix total for ten reactions plus 10% is 2.75 µL, which is above a 1 µL minimum, so the filled-in example is not marked. Two reactions plus 10% is 0.55 µL and is marked. Dilute that stock when the total is below the line.

Scaling template by the overage when it is added per tube. The spare mix is not a spare reaction until you decide to use it. This page counts template tubes from the reaction number alone.

Copying a 25 µL column from the cited protocol while leaving polymerase at 1.25 units. That column is 0.125 µL of a 5 U/µL stock, which is 0.625 units. Units per reaction do not shrink just because the tube is smaller, unless you change the units field.

FAQ

Sources

• Source for the filled-in volumes, not a product endorsement: NEB protocol for Taq DNA Polymerase with Standard Taq Buffer (M0273). 50 µL column: 5 µL of 10X buffer, 1 µL of 10 mM dNTPs, 1 µL of each 10 µM primer, 0.25 µL polymerase (1.25 units), water to 50 µL. Follow your own enzyme manual if it differs.
• Primer Tm for the annealing temperature. This page does not calculate it.
• C1V1 uses the same stock-to-final volume relation for one dilution step.