If you’re running a PCR, you’ll need to make up a PCR mix for each sample. The PCR mix will contain all of the components needed to amplify your target DNA, excluding the DNA template from your sample.
But this doesn’t mean that you need to make up each PCR individually. In fact, it’s much better to make up a batch PCR mix for all of your PCRs in one go. You can then split this mixture across multiple PCR tubes, and add a DNA extract or template for each sample to one of these tubes.
In this article, we explain:
- Why batch PCR mixes are more efficient than making up each reaction separately
- What goes into a batch PCR mix
- Calculating the volumes needed for a single PCR mix
- How to multiply these volumes up for a batch PCR mix for a given number of samples
Why make a batch PCR mix?
When running PCR on multiple DNA samples, it is more efficient to prepare a single batch mix and divide it between tubes, rather than making up each reaction mix separately.
An analogy from baking may help to illustrate the approach: instead of measuring ingredients and mixing batter for each individual cupcake, most people prepare one large bowl of batter and then divide it into individual cupcake cases. This approach saves time, reduces errors, and uses fewer tools.
In the same way, preparing a PCR batch mix simplifies your workflow, ensures consistent reactions, and uses far fewer pipette tips than preparing each PCR individually.
What Goes Into a PCR Mix?
A standard PCR reaction typically includes:
- PCR Master Mix
This is a concentrated solution (e.g. 5x or 2x) containing:- DNA polymerase enzyme
- Reaction buffer
- Magnesium chloride
- dNTPs (DNA building blocks)
- Stabilizers or preservatives
- (Optional) PCR enhancers
- (Optional) Loading dye, containing tracking dyes (often blue and yellow), and a density increasing compound (e.g. Ficoll or glycerol)
- PCR-Grade Water
Ultrapure water, free of DNA, nucleases, and contaminants - Primers
Short DNA sequences that define the region to be amplified - DNA Extract
The sample DNA you want to amplify
Premade Master Mixes contain five or more different components, and so using them can enormously simplify preparation by reducing the number of separate components that you need to add.
Some premade master mixes do not contain loading dyes (these are colourless, while those with loading dyes are coloured). If you are using a premade master mix that’s colourless then you’ll need to add a separate loading dye before loading it into an agarose electrophoresis gel.
Understanding the Units
Volumes in PCRs are usually measured in microlitres (µL), where 1 µL is one-thousandth of a millilitre (mL).
Concentrations in PCRs are given in micromolar (µM) units. This is a standard unit that is based on the number of DNA molecules per litre. If you want to learn more about molarities you can read more on Wikipedia here. While understanding molar concentrations is helpful, most PCR protocols provide recommended volumes of reagents at standard concentrations, so you usually won’t need to calculate concentrations yourself.
Calculating a Standard 20 µL PCR Mix
A common total volume for a PCR reaction is 20 µL. This size is large enough for easy handling and small enough to keep reagent use economical.
Example Components for 1 PCR Reaction
| Component | Volume |
| PCR-Grade Water | 13.5 µL |
| 5x PCR Master Mix | 4 µL |
| Primer Mix (containing two or more primers) | 0.5 µL |
| DNA Extract | 2 µL |
| Total Volume | 20 µL |
Rationale behind the volumes used
- Master Mix: At 5× concentration, use 1/5 the total PCR volume to dilute to 1× concentration (20 µL ÷ 5 = 4 µL of 5x Master Mix per 20 µL PCR).
- Primer Mix: PCRs use a final concentration of 0.1-1 µM of primers. 0.5 µL of an ~0.8 µM primer mix in a 20 uL PCR will give you a 0.2 µM final concentration, which typically performs well without encouraging primer-dimer formation.
- DNA Extract: 2 µL is the minimum volume for accurate pipetting with a 20 µL pipette; more may work better with purified DNA but may introduce inhibitors if the DNA is crude
- PCR Grade Water: Add enough to bring the total to 20 µL (20 – 4 – 0.5 – 2 = 13.5 µL of water per PCR)
Modifications
If you are using individual primers rather than a primer mix, you should add enough of each primer to make around 0.2 µM final concentration, and reduce the amount of PCR-grade water to compensate for the additional volume.
If you want to add any PCR additives, or use more or less DNA extract or purified DNA, you should also subtract or add the appropriate amount of PCR-grade water to make up the final volume to 20 µL.
Making a batch PCR mix
To make a batch PCR mix, just write down what you need for one reaction, taking into account any modifications you need to make for multiple primers or any PCR additives. Then multiply each component by the number of samples you want to do, and then multiply by 1.1 to get an extra 10% to account for any pipetting inaccuracies. The table below shows an example for PCRs using Bento Lab’s primer mixes.
Example components for batch PCR mixes
| Reagent | 1 Reaction | 10 Reactions (+10%) | 20 Reactions (+10%) | 32 Reactions (+10%) |
| PCR-Grade Water | 13.5 µL | 148.5 µL | 297 µL | 475.2 µL |
| 5x Master Mix | 4 µL | 44 µL | 88 µL | 140.8 µL |
| Primer Mix | 0.5 µL | 5.5 µL | 11 µL | 17.6 µL |
| DNA Extract* | 2 µL | 22 µL | 44 µL | 70.4 µL |
| Total Volume | 20 µL | 220 µL | 440 µL | 704 µL |
*Important: Do not include the DNA extract in the batch mix. Add it individually to each tube just before cycling.
Practical Steps
Once you have the list of what to add together, and how much of each component you need, the process is quite straightforward:
- Label your PCR tubes: A simple numbering system of 1–10 or 1–32 can make labelling quicker and easier.
- Prepare the batch mix: Combine the PCR-grade water, 5x Master Mix, and primers in a clean tube, in the volumes required to make your desired number of PCRs. Adding the largest volume reagent first can help you order the reagents and improve pipetting accuracy (it’s easier to pipette small volumes into a larger volume of liquid than into an empty tube).
- Aliquot the PCR mix: Dispense the appropriate amount of PCR mix, excluding the volume left for the DNA extract or DNA template, into each PCR tube. For example, pipette 18 µL into each tube if you are using 2 µL of DNA extract.
- Add DNA extract: Add the DNA sample individually to each tube.
- Seal and cycle: Place the tubes in the thermocycler and start your PCR program.
Additional Tips
Only use PCR-grade reagents and consumables to avoid contamination or inhibition.
Use the most precise and accurate pipette available for the volume you’re handling. For example, it would be more accurate to pipette 35 µL by using a 20 µL pipette twice, rather than a single 35 µL pipetting action with a 200 µL pipette.
If you can’t set your pipette to an exact amount (e.g. 13.6 µL), round to the nearest pipettable amount (e.g. 13.5 µL).
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