Phenol Chloroform DNA Extraction: Basics, Preparation of Chemicals and Protocol

Phenol, chloroform and isoamyl alcohol are three basic and important ingredients used in the present method which relies on DNA precipitation using alcohol. It is abbreviated as the PCI DNA extraction method. 


As these three chemicals are the foundation of the process it is also referred to as phenol DNA extraction, phenol-chloroform or phenol-chloroform isoamyl alcohol DNA extraction. any name we can use. 

For years, the method is widely used, though, is manual but most accurate and scientists love to use it. If you want to know more about the history of DNA extraction, you can read our previous article, here is the link,

Technically, we can categorize it in liquid-liquid DNA isolation, as the main ingredients are liquid. It separates molecules based on their solubility which is indeed an important property of any biological molecule. The separation is done in an immiscible solution.  

The process to isolate DNA from a cell is called “DNA extraction” or “DNA isolation”, various techniques exist each of which has its unique advantages. Proteinase K method, spin- column-based method and CTAB method are several other common DNA isolation techniques, besides phenol-chloroform and isoamyl alcohol.  

Gene amplification, DNA sequencing, restriction digestion and gene quantification are several common applications that rely on the extracted DNA. Conclusively, we need DNA when experiments in genetics.  

In the present article, I will explain the PCI method of DNA extraction, its process, principle, advantages and disadvantages. Besides, I will share some tips and my guide to using it. The article also contains information on how to prepare the phenol, preparation of different solutions & chemicals and so many other things.  

Principle of PCI method:

As we said earlier, phenol-chloroform isoamyl alcohol relies on the principle of liquid-liquid extraction of biomolecules. It denatures the protein portion of a cell and removes it followed by separating genomic DNA into a soluble phase. 

To understand it precisely, we need to look inside the tube, let dive into the tube. 

Suppose the tube is filled with phenol, chloroform, isoamyl alcohol and cell suspension. The phenol is non-polar while the watery part (containing chloroform) is polar in nature. Also, note that phenol is denser than water. The DNA is a non-polar molecule having a negative charge while the protein is a polar (and non-polar) molecule. 

(Some amino acids have polar groups while some have non-polar.) 

The principle of the polarity of biomolecules says that the polar molecules dissolve in the polar solvent and the non-polar molecules in the non-polar solution. 

Henceforth, chloroform dissolves DNA but not protein while phenol digest protein but not DNA. Due to the higher density of phenol, it remains on the bottom. So The upper watery part has genomic DNA while the lower phenol part has digested proteins.

The use of chloroform and isoamyl alcohol adds more benefits to the process hence used. When we centrifuge the tube, a protein with phenol remains at the bottom and DNA at the top. 

This is the simplest explanation of the principle. Importantly during the process, emulsification happens, meaning a foam-like emulsion forms which should be removed first. Note this point, I will explain this part (how to remove foam) later.

Role of each chemical: 

The technique becomes more aggressive when the isoamyl alcohol is used along with phenol and chloroform therefore the technique is often known as PCI DNA extraction. The in-depth role of three major constituents is explained here. 

Phenol:

DNA is insoluble in phenol because phenol is a non-polar solution.

On the other side, protein has both polar and non-polar groups present in it because of the long chain of different amino acids. The amino acid side chains have varied groups.

Also, the folding of the protein into the secondary, tertiary and quaternary structure relies on the polarity of the amino acids. When we add phenol, bonds between amino acids break leads to protein denaturation. 

We can say, phenol unfolds the protein structure and digest it. 

Chloroform:

The main function of chloroform is to protect genomic DNA during a catastrophe. 

Chloroform increases the efficiency of phenol to denature the protein. Here, chloroform allows proper separation of the organic phase and aqueous phase and keeps DNA protected into the aqueous phase.

Note that, chloroform denatures the lipid as well.

Isoamyl alcohol:

Remember the foaming during phenol mixing?

In the phenol-chloroform DNA extraction method, Isoamyl alcohol helps in reducing foaming between interphase. It prevents the emulsification of a solution. 

The liquid phase contains DNA and the organic phase contains lipid, proteins and other impurities. The precipitated protein denatured and coagulated between both these phases. This will create the cloudy, whitish- foam between interphase.

The anti-foaming agent, isoamyl alcohol stabilized the interphase by removing the foaming and increases the purity of DNA. 

Noteworthy, the isoamyl alcohol is also practiced as a DNA precipitation agent in the later stage of the extraction process.  Briefly, the role of each chemical is explained in the table below,

Chemical

Role in DNA extraction

Tris

It maintains the pH of the solution and also permeabilizes the cell membrane.

EDTA

It is a chelating agent and blocks the activity of the DNase enzyme.

SDS

It is an anionic detergent that helps in the denaturation of cell membrane protein.

NaCl

Prevents the DNA denaturation

MgCl2

Protects DNA from mixing with other cell organelles

TE buffer

dissolves DNA

Preparation of phenol: 

We cannot use phenol directly, we have to prepare saturated phenol before proceeding further. The commercially available phenol comes in crystalline form, we have to saturate it before use.

I have performed many DNA extractions and prepared phenol a thousand times. Here is my protocol to prepare the saturated phenol and you can use it. 

Saturation of phenol:

Take the bottle of phenol from the deep freezer and put it in room temperate for some time. After that put the bottle of phenol in the 65°C water bath.

Thaw it at 65°C until the phenol becomes liquid.

Now take the required amount of phenol into a flask and add 0.1% W/V 8- hydroxyquinoline.

Add an equal volume of 0.5M Tris-HCl at pH 8.0

Put the flask of phenol on the magnetic stirrer for 20 to 25 minutes. Stir it and mix well.

Transfer the mixture of phenol and Tris HCl into the separating funnel and leave it for 10 to12 hours for separation.

After 12 hours we get the organic phase and aqueous phase. Collect the lower phase (organic phenol phase) and check the pH with a pH strip. Set the pH between 7.8 to 8.0.

Repeat all the steps until you get the phenol with a pH of 7.0 to 7.5. From the second cycle onward use 0.1M Tris instead of O.5M Tris.

After completion of phenol equilibration, collect phenol in a red amber bottle and add a pinch of 0.2% W/V beta-mercaptoethanol.

Phenol chloroform DNA extraction: Basics, preparation of chemicals and protocol
Image of separating funnel which separates aqueous and organic phases.

Overlay 1 cm of 0.1M Tris into the bottle to protect the phenol (it is light sensitive) and store it at 4°C. Don’t use phenol if pH is changed.

During phenol preparation wear gloves and an eye protector, don’t expose phenol to sunlight. Phenol is volatile and can burn your skin, so handle it carefully.

Preparation of phenol is an important step in DNA extraction, if you prepared phenol well, you will get a good result. Soon after, we need to prepare a solution of phenol: chloroform: isoamyl alcohol.

Preparation of phenol-chloroform isoamyl alcohol

The magic of PCI DNA isolation relies on the use of chemicals, meaning, how and in which amount you use the three ingredients. You will get excellent results if all ingredients are correctly weighed and used. 

The concentration of chloroform and isoamyl alcohol is as important as phenol. The phenol can be used in combination with chloroform and isoamyl alcohol in three different steps,

  • In the very first step use only phenol
  • In the next step use phenol: chloroform: isoamyl alcohol (25: 24:1)
  • In the last step use Chloroform: isoamyl alcohol (24:1)

For 25: 24:1 preparation of PCI, take 25 ml of phenol, 24 ml of chloroform and 1 ml of isoamyl alcohol and mix it well. For 24:1 chloroform: isoamyl alcohol adds 48 ml of chloroform and 2 ml of isoamyl alcohol for 50 ml.

Note that to achieve excellent results prepare each solution fresh every time when doing DNA extraction. Also, prepare it as per your requirement, if you need 10 ml, weigh each ingredient accordingly.

Different types of DNA extraction method
Precipitated DNA

Protocol of Phenol-chloroform and isoamyl alcohol:

The detailed protocol is explained here and this is one of the best standard protocols of our lab.

Collect 5 ml of blood and add 5 ml of solution-I (equal volume) and add 120 µl of Nonidet P40, gently mix by inverting several times until Nonidet P40 mixed in solution, centrifuge at 2000 rpm for 20 min.

For more detail, on solution-I and solution-II, download our eBook on DNA extraction. We will publish it soon.

Discard the supernatant and add 800 µl of solution-II, the sample should be handled gently.

Transfer it in a 2 ml Eppendorf tube, now add 400 µl saturated phenol, mix well and centrifuge at 12000rpm for 1 min.

Take supernatant and add 800 µl of phenol: chloroform: isoamyl alcohol (25:24:1) (400 µl saturated phenol: 384 µl chloroform: 16 µl isoamyl alcohol ) mix well and centrifuge at 12000rpm for 1min.

Take supernatant and add 700 µl of chloroform: isoamyl alcohol (24:1) (672µl Chloroform: 28µl Isoamyl alcohol), mix well and centrifuge at 12000 rpm for 1 min.

Take supernatant and add a double volume of chilled ethanol or add 1/10 vol. of sodium acetate and the equal volume of isopropanol (chilled).

Mix well by inverting until DNA precipitate appears.

Centrifuge at 12000rpm, remove the supernatant and wash DNA with 70% ethanol. Minimum 2 and maximum 5 wash should be given to DNA so that we can get pure DNA.

After the final wash discards the alcohol and dries the pellet overnight or in the hot air oven for 15min 50°C.

Add 100 to 500 µl of dd/w or in TE buffer depends upon the quantity of pellet. Now transfer the Eppendorf tube in the water bath at 65 to 70°C temperature for 30 min or until DNA dissolve.

The given protocol is for 5 ml of a blood sample and it is standardized by our team. You can use it directly. Also, you can modify it as per your sample quantity.

Advantages: 

  • One of the most trusted, well-known and widely accepted methods of DNA extraction is our PCI method. 
  • We get good DNA purity and yields
  • The present method is cheap, easy to use and reliable.
Phenol chloroform DNA extraction: Basics, preparation of chemicals and protocol
DNA extraction experiment of Stephanie Bougel and Jean Benhatter

Stephanie Bougel and Jean Benhatter had used different methods for extracting DNA from 10 unrelated samples. As shown in the graph, Among all 10 samples, the PCI gives a higher yield of DNA in comparison with the Maxwell 16 method. 

However, the yield is lower as compared with Qiagen DNeasy Blood and tissue kit. Still, the amount of DNA obtained from the phenol-chloroform DNA extraction method is good. Read the full research paper of Stephanie Bougel and Jean Benhatter, click here.

Limitations: 

  • The phenol-chloroform method of DNA extraction is time-consuming and tedious. However, by standardizing it properly, we can use it routinely.
  • Also, the process of chemical preparation is time-consuming and tedious too.
  • The chemicals used in phenol-chloroform DNA extraction are dangerous for health which is the major limitation of the PCI method.
  • The phenol is volatile and may cause skin burn and irritation.
  • The chloroform makes you unconscious. 
  • Moreover, what you get depends on how you work, meaning if you don’t have a good practical hand, you can’t isolate good DNA.
Phenol chloroform DNA extraction: Basics, preparation of chemicals and protocol
DNA extraction experiment of Natalia Gumińska et al.,

Natalia Gumińska et al. had been used the DNeasy Plant DNA extraction kit, DNeasy Blood and Tissue DNA extraction kit, phenol-chloroform method, CTAb method to extract DNA from various sources. 

As per their findings, the purity and quantity obtained from the phenol-chloroform DNA extraction method were very less. The 260/280 ratio of the PCI method wasn’t so good and the quantity too not so sufficient. 

The graphical representation of their findings is shown in the figure above. You can read their article here, click here. 

 In summary, the purity and quantity obtained by the phenol-chloroform isoamyl method aren’t so good. 

My suggestions: 

Safety is a priority, chemicals used during DNA extraction can be harmful in many ways therefore always wear gloves, eye protector, head cap and face mask.

While handling phenol, always wear a lab coat and eye protector because phenol is volatile and can burn your skin. It can also damage our eyes hence do not compromise safety.

The chloroform can make you faint or unconscious, a high dose can be lethal so take enough precautionary steps before performing. 

Moreover, we need to protect our chemicals and solutions too. For instance, phenol can oxidize when exposed to sunlight therefore always store phenol in a dark or amber bottle.

Also, the pH can be fluctuated at a higher temperature so always store phenol at 4°C, check the pH periodically.

Prepare fresh phenol: chloroform: isoamyl alcohol every time before DNA extraction.

We have explained each step, the composition of each chemical, solution I and solution II in our book. Also, we have described the protocol for 200 microliters of sample. Purchase it when published.

If you wish to learn more, want to use more protocols and standardize methods, buy our ebook here: From DNA extraction to PCR.  

Set up your own DNA extraction lab by reading this article:

Conclusion:

For a startup or a new lab, the present method (Phenol-chloroform and isoamyl DNA extraction) is the best option- cheap, effective and reliable. It cuts costs and gives excellent results too if performed well. 

I personally believe that every student should learn manual DNA extraction, use of chemicals, solution preparation and protocol standardization. Although ready-to-use kits are now common, try to learn manual things, thrust me it makes your practical hand more efficient.  

Always prefer to do DNA extraction with your own combination of chemicals, it will boost your knowledge and confidence.

12 thoughts on “Phenol Chloroform DNA Extraction: Basics, Preparation of Chemicals and Protocol”

  1. Abhishek Gupta

    i have prepare the mixture of phenol cgloroform and isoamylalcohal in 24: 24 :1 ratio but after mixing phenol into chloroform isoamyl alcohal solution it forms 2 phases seperately. can you please guide ?

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