“PCR master mix is a pre-mix that includes all the essential PCR ingredients except template DNA and primers. Learn its importance and how to prepare it in your lab.”
Polymerase chain reaction plays a crucial role in molecular genetics. Since most experiments highly rely on amplification, PCR holds unmatched importance. It’s been used in all life science fields.
It is a multi-step process. Every step has its own challenges. However, the success of the experiment highly depends on the reaction preparation, which is a difficult and error-prone step. Using a pre-PCR mix can help avoid these mistakes.
In this article, we will discuss the PCR premix or master mix, its components and importance, most importantly, how you can prepare it in your lab.
Stay tuned.
Key Topics:
What is a PCR master mix?
PCR master mix is a premix of important PCR ingredients, including dNTPs, buffer, Taq DNA polymerase, and other PCR enhancers. It is available commercially and can also be prepared in-house.
Students often fail to get consistent results due to reaction-handling errors. For beginner’s it is a common PCR error, adding 5 to 7 ingredients in 5 to 50 tubes is a difficult task, indeed, and likely to fail the reaction.
In addition, the concentration of each ingredient plays a crucial role in PCR success and hence, care must be taken while preparing the PCR master mix.
Using the master mix reduces both the error rate and contamination, as most of the ingredients have been added from a single solution with just a single pipette tip.
PCR reagents:
dNTPs, PCR buffer, Taq DNA polymerase, primers, template DNA and nuclease-free water are common PCR ingredients. Their function and concentration are explained in the table below.
| Ingredient | Function | Concentration |
| dNTPs | Building blocks for a new DNA strand | 200 µM each (dATP, dTTP, dCTP, dGTP) |
| Taq DNA polymerase | Catalyzes DNA synthesis by extending primers | 0.5–1.25 U per 50 µL reaction |
| PCR buffer | Maintains optimal pH and salt for polymerase | 1X |
| Primers | Binds with the DNA strand | 10 pM |
| Template DNA | Provides the target sequence to be amplified | 1–10 ng (plasmid) or 10–100 ng (genomic) |
| Nuclease-free water | Brings the total volume to the desired reaction size | – |
| MgCl2 | Cofactor for DNA polymerase activity | 1.5–2.5 mM |
Why do we need to prepare a PCR master mix?
Manual PCR reaction preparation has its limitations, therefore, a master mix is needed.
- Adding ingredients individually is a tedious and time-consuming process.
- It’s also highly error-prone—students may accidentally skip an ingredient, mix up components, or add the wrong volume.
- Contamination chances are always high during PCR. For high-throughput analysis, students can make mistakes while handling the reaction or pipetting, leading to false-positive or false-negative results.
- It delays reaction and thus, non-specific amplifications and primer dimers are possibly observed due to early activities of the ingredients.
- It also increases the chances of reaction failure. Due to prolonged exposure to the room temperature, ingredients like dNTPs, Taq DNA polymerase or buffers lose their strength.
Why is preparing a PCR master mix beneficial?
Preparing a PCR master mix is beneficial in many ways to students.
- First, it saves time; extensive and repeated pipetting is not required.
- It reduces the chances of reaction failure as all the ingredients are added from a single pipetting step.
- It has the lowest reaction contamination chances as repeated working or stock solution preparation is not needed.
- It minimizes the reaction exposure to the environment, thereby reducing the chances of non-specific amplification.
- It is suitable for multiplex and high-throughput analysis.
DIY: How to prepare 10X PCR master mix:
First, check out what to include and what not to include in the master mix.
| Component | Include/ not-include |
| dNTPs | ✔️ |
| Buffer | ✔️ |
| Taq DNA polymerase | ✔️ (not recommended) |
| MgCl2 | ✔️ |
| KCl | ✔️ |
| Nuclease-free water | ✔️ |
| Forward primer | ❌ |
| Reverse primer | ❌ |
| Template DNA | ❌ |
The PCR buffer and enhancer components, like the Tris-HCl, KCl, MgCl2, dTNP mix, Taq DNA polymerase and nuclease-free water, remain the same for all the reactions.
The template DNA and primer set may vary among samples and DNA, respectively. Therefore, the PCR master mix can be prepared by mixing all the essential common ingredients and excluding primers and template DNA.
It is recommended to prepare a stock master mix of 10X to avoid repeated preparation. You can prepare the stock and make the working solution as per the requirement.
Always remember one thing while preparing stock solutions: you have to back-calculate the concentrations.
To understand this in a much better way, let’s understand the table below, where standard concentrations of 1X PCR master mix are given, and for most reactions, this is the ideal concentration.
| Reagent | Final conc. in 1X | Required in 10X |
| Buffer | 10 mM | ?? |
| KCl | 50 mM | ?? |
| MgCl₂ | 1.5 mM | ?? |
| dNTPs (each) | 0.2 mM | ?? |
| Taq Polymerase | ~0.025 U/µL | ?? |
Now, we have the ideal 1X concentration for the 25 µL reaction. Using this reference value, we will back-calculate the concentration for the 10X stock solution.
Confusing?
Let’s take an example of MgCl2. You need 1.5mM in your final reaction mixture of 25 µL. You have to prepare a 10 times more concentrated stock solution, i.e., 10X.
1X → 10X, 1.5 mM → 15 mM
So you will have to prepare the 10X master mix where the concentration of MgCl2 will be 15 mM.
You can also calculate this by the equation N₁V₁ = N₂V₂, where:
- N₁ = concentration of stock solution
- V₁ = volume of stock solution to add
- N₂ = desired final concentration
- V₂ = final total volume
Let’s say you want 1.5 mM MgCl₂ in your final PCR reaction (25 µL), and you’re using a 10X master mix – meaning you’ll use 2.5 µL (As dilution is 1:10, so 25/10) of 10X master mix in the reaction.
So, N1= ??,
V1=2.5 µL,
N2=1.5 mM and
V2=25 µL.
Let’s put these values in the equation,
⇒N1 × 2.5 µL =1.5 mM × 25 µL
⇒V1 = 1.5 mM × 25 µL / 2.5 µL
⇒N1 = 15 mM
Let’s say you want 1.5 mM MgCl₂ in your final PCR reaction (25 µL), and you’re using a 2X master mix — meaning you’ll use 12.5 µL of 2X master mix in the reaction.
So, N1= ??, V1=12.5 µL, N2=1.5mM and V2=25 µL. Let’s put these values in the equation,
⇒N1 × 12.5 µL =1.5 mM × 25 µL
⇒V1 = 1.5 mM × 25µL / 12.5µL
⇒N1 = 1.5 mM × 2 = 3.0 mM
So, in 10X master mix concentration of MgCl2 will be 15 mM, but in 2X master mix it will be 3 mM, and in 1X master mix it will be 1.5 mM. So this way you can adjust the concentration of MgCl2 between 1.5 mM to 2.5 mM.
Similarly, you can calculate the concentrations of other reagents such as dNTPs, buffer, and KCl. You will get the concentrations of each reagent for the 10X PCR master mix as mentioned in the table below.
| Reagent | Final conc. in 1X | Final conc. in 2X | Required in 10X |
| Buffer | 10 mM | 20 mM | 100 mM |
| KCl | 50 mM | 100 mM | 500 mM |
| MgCl₂ | 1.5 mM | 3 mM | 15 mM |
| dNTPs (each) | 0.2 mM | 0.4 mM | 2 mM |
| Taq Polymerase | 0.025-0.05 U/µL | 0.05-0.1 U/µL | 5 U/µL |
Once you prepare the 10X stock master mix, you have to dilute it into 2X for routine PCR reaction setup of 25 µL.
So, without wasting time, let’s prepare the direct 2X working PCR master mix.
2X PCR master mix:
To make a 2X PCR master mix, you have to dilute the 10X stock solution 5 times. For example, you want to make 100 µL of 2X PCR master mix from a 10X stock solution.
Then take 20 µL from 10X PCR master mix and add 80 µL nuclease-free water to make 100 µL 2X PCR master mix.
You can also calculate this by the equation N1V1=N2V2,
Where N1= 10X, V1= ??, N2= 2X, and V2= 100 µL.
So V1= (N2 × V2) / N1 = (2X × 100µL) / 10X = 20µL
Please note that I have taken the volume of 2X master mix as 100 µL, just for example, you can take whatever amount you require.
Let’s prepare it practically. This preparation is assumed to make 100 µL 2X PCR master mix from 10X stock master mix solution, However, you can make whatever amount you require according to the sample size you want to process for PCR.
- Prepare your working bench or biosafety cabinet.
- Prepare yourself following the standard lab safety and contamination protocol.
- Collect all the reagent tubes for the deep freezer.
- Place it on the ice and start thawing reagents one by one.
- Now, take a clean and sterile microcentrifuge tube.
- Take 20 µL of 10X master mix and add 80 µL nuclease-free water to make 100 µL, working 2X PCR master mix.
- Our 2X PCR master mix is ready to use.
Now take 12.5 µL 2X master mix directly for the reaction preparation, 5.5 µL nuclease-free water, and 1 µL of each primer to make a reaction mixture of 20 µL, and then add 5 µL of template DNA to make a 25 µL reaction setup.
Types of PCR master mix
In this section, I will review several different types of commercially available master mixes and applications.
Complete master mix:
The complete master mix contains all the ingredients, including the buffer, Taq DNA polymerase, dNTPs, and PCR enhancers. It’s a kind of one-go mix widely used in the molecular lab.
Master mix without Taq DNA polymerase:
Taq DNA polymerase is the most sensitive ingredient among all these. Adding Taq separately as per the requirement is sometimes needed. Some scientists prefer to use this type of master mix.
Master mix with a dye:
Master mix with a SYBR green electrophoresis dye is widely popular. It is green in color and already contains the electrophoresis dye and all other PCR ingredients.
dNTP mix:
Some scientists and laboratories prefer only the dNTP mix and buffer, and Taq DNA polymerase separately. This increases the customization options and is mostly used to optimize an assay.
Wrapping up:
So this is a complete and proven recipe to prepare the working PCR master mix. It works for the majority of the experiments. However, for several specialized experiments, additional optimization is required.
To strengthen your knowledge, you can read the previous article on our PCR resources. I hope you like this article. Do share and subscribe to Genetic Education.
