Preparing the PCR reaction is one of the important steps in PCR amplification. Taq DNA polymerase, dNTPs, PCR reaction buffer, PCR primer and template DNA are important ingredients of PCR reaction.

Preparing a perfect PCR reaction is key to success amplification.

Not only amplification but a specific amplification is a goal for our PCR reaction. We don’t want unwanted DNA bands and Primer dimers.

The primer dimers are the foe for the PCR reaction.

W can divide our PCR reagents into three categories.

Category 1: Very important (core) ingredients: dNTPs, primer, template DNA and Taq DNA polymerase

Category 2: Crucial (subsidiary) ingredients: PCR reaction buffer, MgCl2, DMSO and other PCR enhancers.

Category 3: Least important: Water and DNA gel loading dye (some of the ready to use mastermix contains the gel loading dye in the mastermix that does not have any significant role in the amplification. We can avoid it or add it later during the gel electrophoresis).

Without category 1 reagents, the PCR reaction cannot happen. We can not amplify DNA.  However, the subsidiary ingredients which are the PCR enhancers are not as important as core ingredients.

But enhancers are very important for the amplification of tough templates such as GC rich template and long DNA template.

Note: The amplification of long template DNA required a special type of PCR setup called a long-range PCR. We had covered an interesting article on the long-range PCR, please read it here: A comprehensive handbook of long-range PCR.

Category 3 PCR ingredients are least important, it does not have any notable impact on the amplification process.

Here in this article, we will discuss 10 secrets that nobody tells you about the perfect PCR reaction.

Key points:

  • The secrets:
  1. Proper DNA extraction
  2. The concentration and the components of a master mix
  3. The types of DNA polymerase
  4. Primer designing
  5. Use of PCR reaction buffer
  6. The concentration of template DNA
  7. The annealing temperature of the reaction
  8. The concentration of MgCl2
  9. PCR cycles
  10. other utilities
  • Preparation of PCR reaction
  • Summary
  • Conclusion


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1.Proper DNA extraction:

The “mantra” for successful PCR amplification depends on how we extract our DNA. No one takes DNA extraction as a serious business.

But trust me if you extract a high quality nearly ~1.80 extra pure DNA, your 50% work for the PCR reaction is almost done.

The impurities present into the template DNA hinders in the amplification called PCR inhibitors. SDS, phenol and proteins are common among them.

No matter, how perfectly you optimize your reaction if your DNA is not properly extracted, your PCR’s future will be in danger.

However, it is possible that our DNA is not perfectly extracted every time. But before use it in the amplification, purify the DNA using the DNA purification kit. Or else re-extract your DNA.

different types of DNA extraction methods

The image represents the general outline of how DNA extracted from the cell.

Ideal concentration of template DNA: 30 to 50ng (for 25 μL reaction)

The purity of DNA: 1.77 to 1.89 ( 260/280 ratio)

For more detail, on the DNA extraction, you can read our previous article: Different types of DNA extraction methods

2. The concentration and the components of mastermix:

The Taq DNA polymerase amplifies the growing DNA strand by incorporating the dNPTs. That means, “no dNTPs no amplification.”

However, the concentration of the dNTPs is very important, if the concentration is too high, the chance of non-specific amplification increases. The reagents are wasted too.

I always prefer to use the ready to use mastermix which contains all the dNTPs (dATP, dGTP, dCTP, dTTP) along with the gel loading dye.

The ready to use mastermix is the best choice, it saves time during PCR reaction preparation as well as during the agarose gel electrophoresis.

PCR reaction: Ten secrets that nobody tells you

However, if you are enthusiastic enough to do it with yourself, that is nice. Because we can optimize the concentration of each dNTPs as per our requirement.

The ideal concentration of each dNTPs in PCR reaction is,

dATP:  200μM

dTTP: 200μM

dGTP: 200μM

dCTP: 200μM

The total concentration of the dNTPs in the PCR reaction must be 800μM for 50μL. For 25μL use 400μM (50μM each dNTPs) in the PCR reaction.

I hope so you don’t require calculations.

3. The types of DNA polymerase:

The DNA polymerase is another important category 1 core ingredient. The DNA polymerase helps in synthesizing the DNA using the Mg2+ as cofactor and dNTPs.

The very first choice for any PCR reaction is the Taq DNA polymerase.

The amplification speed of the Taq DNA polymerase is very high, it is thermostable and can work efficiently even at a higher temperature.

When we are talking about the standard PCR reaction, the Taq is fine, however, it is not suitable for all type of amplification reactions.

If the specificity is a prime goal of your PCR reaction, use Hot start DNA polymerase.

If proofreading is a prime goal of your PCR reaction than use high fidelity DNA polymerase. The high fidelity DNA polymerase amplifies DNA as well as removes the mismatched nucleotides from the growing DNA strand.

DNA polymerase Use for Concentration
Taq DNA polymerase Shorter and conventional amplification. 1U
A hot start DNA polymerase For diagnostics and low abundant DNA templates. 0.8- 1.0U
A high fidelity DNA polymerase DNA sequencing, Site-directed mutagenesis and DNA microarray. 1U

For more detail on DNA polymerase, Taq, hot start DNA high fidelity DNA polymerase please read this article: Choosing the right DNA polymerase for your PCR experiment.

4. Primer designing:

The primer is a short single-stranded DNA used to amplify the template DNA. The primer sequence is complementary to the target region of our DNA.

It provides the free 3′ end for the addition of dNTPs for Taq DNA polymerase.

Designing the primer is an important agent in the PCR dry lab work. The primer 3 is the best choice for designing the primer for any PCR reaction.

It provides all the information about how the primer amplifies and behaves during the amplification of a DNA.

PCR reaction: Ten secrets that nobody tells you

The Taq DNA polymerase amplifies DNA once it finds the OH group of primer.

The ideal primer should have the following properties:

  •  Contains 50 to 55% of GC content
  • At least 18 to 22 nucleotides long
  • Does not contain secondary structure
  • The annealing temperature between 50°C to 65°C

The primer contains all these properties is a champion of our PCR reaction however, likewise, one criterion is as important as the above listed.

The concentration of the primers.

The stock primers are supplied under various concentrations we have to decide which concentration works best for our PCR reaction.

If the concentration of the primer is higher than the desired one, primer dimers and non-specific amplification occurs during the PCR reaction.

On the other side, if the concentration of the primer is too low, it can not amplify even the DNA of our interest.

So, the concentration of primer is a decisive to do the amplification.

The ideal concentration of the primer is,

Primers Concentration
Forward primer 10pM
Reverse primer 10pM

10 pM is enough for our PCR, Although I had obtained PCR amplification with 7 pM primers, without non-specific bands and primer dimers.

Yet, the concentration of the primer more than this creates an unnecessary problem for the PCR reaction. still, you have to optimize your own primer concentration using the gradient PCR machine.

Read more on the PCR primer designing: PCR primer design guidelines.

5. Use of PCR reaction buffer:

Adding the reaction buffer in each and every reaction is now an obvious process in the PCR reaction, but it is not a good practise, we do not need PCR reaction buffer for all the PCRs.

Yes, it is true. Do not use PCR buffer in all reactions, it is just a waste of money. For a simple and conventional PCR, the core ingredients work finely.

The PCR reaction buffer is the combination of different chemicals which enhance the amplification efficiency of the reaction.

Generally, the PCR reaction contains MgCl2, DMSO, KCl, (NH2)SO4, albumin, betanins and other secret ingredients.

Actually, it provides flexibility in the amplification. So non-specific bindings and primer dimer are easily formed by using the PCR reaction buffer.

So when to use PCR reaction buffer?

You should know when to use the PCR buffer, when it required and how to use it.

The PCR reaction buffer is required in the amplification of the longer templates, high GC rich template and for the sensitive templates.

If your target DNA is too long you have to use the PCR enhancers to amplify it efficiently.

If your PCR template DNA is high GC rich, you need some extra ingredients to push the amplification at its best.

Notwithstanding this, for each and every reaction you must have to optimize using the PCR buffer and without PCR buffer if it is “OK” without extra ingredients than go for it.

Why should waste money and chemicals? if it works without extra ingredients?

We had covered three articles on how different types of PCR enhancers work during the PCR reaction.

The list of the articles are:

Tips 1: If you are using a PCR buffer in a reaction and observed some non-specific bands and primer-dimers, try the reaction next time without using the PCR reaction buffer.

You will definitely get good results.

Tips 2: For more than one set of primers ( multiplex reaction and internal control) you must include the PCR reaction buffer in it.

Read more on multiplexing: Multiplex PCR.

6. The concentration of template DNA:

Before doing the PCR, you have to ask yourself, what problem can be occurred during the PCR?

  • Non-specific bindings
  • Primer Dimers
  • No-amplification

If you do not get any amplification, that is totally a different problem, we will discuss it later.

The primer dimer and non-specific bindings are actually a real problem which zones the PCR amplification.

The concentration of the template also plays an essential role for efficient amplification of DNA.

If the concentration of DNA is too high, it facilitates non-specific bindings.

Template DNA   Concentration Purity
For 25 μl reaction 30 to 50 ng ~1.77- 1.88
For 50 μl reaction 50 to 100 ng ~1.77- 1.88

I always prefer to use 30ng DNA in the 25 μl reaction and it works totally fine for my PCR reaction. De facto, the concentration lower than this can amplify the DNA appropriately. 

PCR reaction: Ten secrets that nobody tells you

A brief overview of Denaturation, Annealing and Extension steps of PCR.

7. The annealing temperature of the reaction:

What is the end product or how your results appear on a gel is totally depends on the annealing temperature of the reaction.

The annealing temperature is a temperature at which the primer binds to its complementary sequence on to our DNA. It is specific for primer.

So if you compromise the annealing temperature, that will create a big problem over a period of time in your amplification and believe me, you can not even resolve it.

If your annealing temperature is too low, you will get so many bands in a gel. If it is too high no amplification observed.

I suggest you to perform a gradient PCR assay for getting an exact annealing temperature even if you have the annealing temperature.

Still, you can trick your annealing temperature by adding ingredients such as MgCl2 and DMSO but it required a high level of expertize.

A pinch of MgCl2 can amplify DNA even at a higher annealing temperature up to 2°C.

But don’t trick the PCR, instead, use the gradient assay and determine your own PCR annealing temperature.

For more detail on the annealing temperature, read this article: PCR primer design guidelines.

8. The concentration of MgCl2:

MgCl2 is one of the pivotal ingredients of the PCR reaction. It boosts the activity of the DNA polymerase.

Every enzyme required a metal ion as a cofactor for performing an enzymatic reaction. The Mg2+ ions of the MgCl2 bind to the active site of the DNA polymerase and activate it.

So you need some extra MgCl2 in the reaction.

Although, the manufacturer already given it in the Taq buffer or in the Taq directly, that is up to the manufacturer. Even some of the companies provide their own buffer for reviving the DNA polymerase which contains the MgCl2.

On the other side, if we are using a ready to use mastermix, it must contain MgCl2 in it.

Read the manufactures guide before using any reagent.

If the MgCl2 is not provided in any of the reagents, use a pinch of it.

Cocnetration of MgCl2: 1.0mM to 2.0 mM (up to 5mM, depending upon the requirement).

Remember, if the concentration of the Mg2+ is too high in the reaction, non-specific bands will appear. So always optimize it before proceeding further.

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9. PCR cycles:

No one customizes it, instead, people are using it by default to 30 or 35 cycles.

But in some cases, or we can say in almost all standard PCR cases there is no need to use PCR for 35 cycles, it wastes the power and consumes only more time.

In addition, using more PCR cycles, the chance of the non-specific bindings are increased along with the primer dimers.

Ask why?

Because after a certain period of timers, the template stops amplifying due to lack of dNTPs, Taq DNA polymerase, template DNA or any other reagent, the amplification is ended but the dimers are continued to expand.

And that creates a problem.

So why to ruin our own PCR. Rather, use fewer numbers of PCR cycles of 22 to 25. If less amplification occurred it will notify you to increase a couple of cycles in the next PCR and that is totally valid.

PCR reaction: Ten secrets that nobody tells you

An overview of the 25 cycles of PCR

Each step in the PCR cycles should be performed perfectly to maximize the yield of the reaction. The numbers of cycles matter a lot in quantitative real-time PCR.

10. Other utilities:

What we had discussed so far is something very important to do while doing the PCR reaction. But other utilities what we are using in the PCR are necessary too.

PCR tubes, Eppendorf tubes and tips used during the reaction preparation plays an active role. The plasticware used during the reaction preparation must be nuclease-free.

The nuclease cuts DNA into smaller pieces. Therefore make sure that the tips and PCR tubes must be nuclease-free.

Further, it must be sterile. if not, autoclave it before doing PCR because if it contains some of the foreign DNA, it might be amplified and falsify the results.

Some of the other contaminants such as traces of other chemicals might hinder in the DNA synthesise, therefore, It is necessary to keep all the utilities clean before preparing the PCR reaction.

The last and the most important bonus tips for doing the excellent amplification is,

Prepare PCR reaction on ice

Using ice is a debatable topic since long for the scientist.

Some say it is fine without the ice. But I want to recommended to use the ice while preparing the PCR reaction (If you are using a normal Taq DNA polymerase).

The Taq DNA polymerase actively involves in the synthesis of DNA even at a lower temperature. Therefore, when you are preparing the reaction, inside your PCR tube, the Taq starts amplifying anything which is complementary to the primers.

This is a major reason for primer-dimer and the non-specific bindings in the PCR.

Although the specificity achieved is high, the primer-dimers still appear in the results due to this reason. If this is the case with you try doing the reaction on ice.

or

Use the hot start Taq DNA polymerase. We had already discussed the hot start DNA polymerase and hot start PCR in two of our articles. Read it here,

  1. What is a hot start PCR?
  2. Choosing the right DNA polymerase for your PCR experiment

Now, let us prepare the PCR reaction.

This section is the hypothetical representation of the PCR reaction preparation.

Preparation of PCR reaction:

In this section, we will explain to you how you can add different components into the PCR reaction and what to do while preparing the reaction.

In the very first step wear gloves, a lab coat, a mouth cap and head cap. Take all the reagents from the fridge and put it on the ice.

PCR reaction: Ten secrets that nobody tells you

 

In the next step, labelled all the PCR tubes properly and thaw the reagents. After that in the very first step add the master mix containing all the dNTPs at the bottom of the tube.

PCR reaction: Ten secrets that nobody tells you

After that, add the template DNA at the bottom of the tube.

PCR reaction: Ten secrets that nobody tells you

After the addition of template DNA, Add PCR reaction buffer to the side of the PCR tube.

PCR reaction: Ten secrets that nobody tells you

Now, add both the primers on the side of the tube. Do not add primers directly in the bottom of the tube because it can

create primer dimers by binding with each other. Remember do not mix both the primers with each other too.

PCR reaction: Ten secrets that nobody tells you

 

In the fifth step add the Taq DNA polymerase to the reaction. Add the polymerase in the last step so that it can not start reaction early.

PCR reaction: Ten secrets that nobody tells you

Finally, in the last and the 6th step add the nuclease-free water to the reaction.

PCR reaction: Ten secrets that nobody tells you

 

Our PCR reaction is now ready. Spin all the tube and put it immediately into the PCR machine.

PCR reaction: Ten secrets that nobody tells you

Summary:

Component Requirement
dNTPs 50mM each
Forward primer 10pM
Reverse primer 10pM
Ta DNA polymerase 1 unit
Template DNA 30ng
Nuclease-free water As per requirement
PCR reaction buffer (optional) 1X
MgCl2 (optional) 2mM

These external resources contain all the information on the PCR,  PCR reaction preparation and PCR protocols:
PCR Protocols PCR Technology

Conclusion:

PCR reaction preparation step is the key to success in the PCR and the successful PCR is the crucial step in the downstream applications such as DNA sequencing and hybridization.

Therefore, PCR reaction should be performed perfectly. The concentration of each and every component used in the PCR is one of the major factors on which your results are dependent.

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