All articles, DNA Extraction, ExplorePhenol chloroform DNA extraction: Basics, preparation of chemicals and protocol

Phenol chloroform DNA extraction: Basics, preparation of chemicals and protocol

Phenol-chloroform DNA extraction method is a well known and widely accepted DNA extraction method since long. We had discussed the history and outline of different types of DNA extraction method in our previous article. 

Please read the article here,

different types of DNA extraction method

Phenol, chloroform and isoamyl alcohol are top three ingredients which is used in liquid-liquid DNA extraction. The solubility is an important property of any biological molecules. Different molecules can be separated on the basis of their solubility in solution or water.

The liquid-liquid DNA extraction method separates DNA molecule on the basis of their solubility in immiscible solutions and this is the basic principle of Phenol chloroform DNA extraction method.

In this article, we will discuss the phenol-chloroform DNA extraction method, preparation of phenol-chloroform and isoamyl alcohol, role of each chemical, preparation of phenol for DNA extraction and finally my ultimate guide for phenol-chloroform DNA extraction method.

The content of the article,

  • The basic principle of phenol-chloroform DNA extraction
  • Role of each chemical in Phenol chloroform DNA extraction
  • Preparation of phenol for DNA extraction (saturated)
  • General protocol of phenol-chloroform DNA extraction method
  • Preparation of each chemical
  • Advantages of phenol-chloroform DNA extraction method
  • Limitations of phenol-chloroform DNA extraction method
  • My ultimate guide for phenol-chloroform DNA extraction method

The basic principle of phenol-chloroform DNA extraction method

The basic principle of phenol-chloroform DNA extraction method is based on the liquid-liquid extraction of biomolecules.

The protein portions of the cell are denatured and removed by separating DNA into soluble phase. The entire mechanism of separation is based on the solubility of the biomolecules.

To understand this mechanism we have to go inside the DNA extraction tube. Let’s get into the tube having phenol and cell suspension.

Water is a polar solvent and phenol is a non-polar solvent. Also, phenol is denser than water. On the other hand, DNA is a polar molecule with a net negative charge on its backbone and protein is non-polar. The entire mechanism of a polarity of DNA is explained in our previous article: Role of alcohol in DNA extraction. Please read the article before going deep into DNA extraction. 

As we all know that the polar molecule can dissolve in polar solutions hence DNA can dissolve in water but not in phenol.

Protein is macromolecule which means it is larger than DNA. Additionally, water can not mix with the phenol so phenol remains at the bottom due to its higher density. Meanwhile, DNA dissolves in water and remains on the top of the phenol as a watery layer, aqueous phase.

When we mix phenol with the cell suspension the protein portion of the cell get digested or denatured and when we centrifuge it, the denatured protein settled into the bottom of the tube along with the phenol.

This has actually happened inside the tube. During the process of mixing, when sample and phenol are mixing togather, it forms the foam like emulsion (emulsification). Now it is important to remove this foam. Note down this point, we will discuss it later: how to remove a foam.

Read further on PCR,

  1. The Function of dNTPs in PCR reaction
  2. Role of DMSO in PCR: DMSO a PCR enhancer
  3. Function of taq DNA polymerase in PCR
  4. PCR primer design guidelines
  5. Role of MgCl2 in PCR reaction

Role of each chemical in Phenol chloroform DNA extraction

The phenol-chloroform DNA method is more effective along with isoamyl alcohol. Therefore sometimes it is also called as PCI or phenol chloroform isoamyl alcohol method of DNA extraction. Role of each chemical 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 group because protein is a long chain of different amino acids. Different amino acids have different groups present on their side chain. 

Also, the folding of the protein into the secondary, tertiary and quaternary structure is depends on the polarity of the amino acids. Collectively, the bonds between amino acids are broken by the addition of phenol and protein is denatured.

Ultimately, we can say the protein become unfolded by addition of phenol.

Chloroform:

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

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 emulsification of solution. 

The liquid phase contains DNA and the organic phase contains lipid 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. This will increase the purity of DNA. 

We had discussed the role of other chemicals used in the phenol-chloroform DNA extraction method in the last article: Different types of DNA extraction methods.

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 permeabilize the cell membrane.

EDTA

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

SDS

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

NaCl

Prevents the denaturation of DNA

MgCl2

Protects DNA from mixing with other cell organelles

TE buffer

It dissolves DNA

Preparation of phenol for phenol Chloroform DNA extraction method

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.

Take the bottle of phenol from the deep freezer and put it at 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 pH strip. Set the pH between 7.8 to 8.0.

Repeat all the step until you get the phenol with 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
image of separating funnel which separates aqueous and organic phases.

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

During phenol preparation wear gloves and eye protection, don’t expose phenol in sunlight. Phenol is volatile and can burn the 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. After the phenol preparation, we have to prepare the solution of phenol: chloroform: isoamyl alcohol.

Preparation of phenol chloroform isoamyl alcohol

PCI method is based on the composition of chemicals, if you prepared each chemical very well and maintained the composition of each chemical, you will get excellent results.

The concentration of chloroform and isoamyl alcohol is as important as phenol. The phenol can be used in combination of 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 add 48 ml of chloroform and 2 ml of isoamyl alcohol for 50 ml.

Freshly prepare each combination of chemicals every time. Hence prepare according to your requirement. If you require only 10 ml of PCI prepare accordingly.

Different
Precipitated DNA

General protocol of phenol-chloroform DNA extraction method

The detailed protocol is explained here and this is one of the best standard protocol 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 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 final wash discard the alcohol and dry 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 

The phenol-chloroform DNA extraction method is traditional and widely accepted in genomic research.

The amount of DNA obtained form PCI method is very high. Also, the quality of DNA is very good as well.

The PCI method of DNA extraction is cheap and reliable.

Phenol
DNA extraction experiment of Stephanie Bougel and Jean Benhatter

Stephanie Bougel and Jean Benhatter used different methods for extracting DNA from 10 unrelated samples. As shown in the graph, the phenol-chloroform method of the DNA extraction yields comparatively high amount of DNA as compared with Maxwell 16 method. 

However, the yield is lower as compared with Qiagen DNeasy Blood and tissue kit. Still, the amount of DNA obtained from phenol-chloroform DNA extraction method is good. You can 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.

The chemicals used in phenol-chloroform DNA extraction are dangerous for health which is the major limitation of the PCI method.

Also the quantity and quality of DNA may vary depending upon the processing hand.

Phenol
DNA extraction experiment of Natalia Gumińska et al.,

Natalia Gumińska et al., explained different methods of DNA extraction by comparing 5 different types of DNA extraction methods. For their study, the selected methods are; DNeasy Plant DNA extraction kit, DNeasy Blood and Tissue (DNA extraction kit), phenol-chloroform method, CTAB traditional method and CTAB modified method of DNA extraction. 

As per their findings, the purity and quantity obtained from the phenol-chloroform DNA extraction method are very less. The 260/280 ration of DNA may vary up to 2.0 which is not good.

Also, the quantity of DNA is less than 100ng/microliter. The graphical representation of their findings are enlisted into the figure above and indicated with the red arrow. For more detail on their findings please read the research article of Natalia Gumińska et al., click here. 

My ultimate guide for phenol-chloroform DNA extraction method

Safety is a priority, you have to take some precautions before proceeding for DNA extraction.

Always wear gloves, goggles, cap and face mask.

While handling phenol, always wear lab coat and goggles because phenol is volatile and can burn the skin. It can also damage our eye hence do not compromise with safety.

The chloroform can make you faint. Also, the higher dose of the chloroform can be life-threatening. So be protective while handling chloroform.

Along with self-precautions we have to take care of each chemical as well. Phenol can oxidize into sunlight therefor always store phenol in dark or amber bottle.

Also, the pH can be fluctuated at a higher temperature so always protect phenol at 4°C. additionally, check the pH of the phenol periodically before proceed.

Prepare phenol: chloroform: isoamyl alcohol fresh 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.

Phenol-chloroform method of DNA extraction is one of the outstanding methods since long. Every student should learn DNA extraction through the phenol-chloroform method. The method is very cheap and cost effective therefore the phenol-chloroform DNA extraction method is the best alternative for those laboratories which are under growing phase.

I personally believe that every student should learn how to use different chemicals and how to modify the protocols for extracting DNA.

Read further,

  1. Agarose gel electrophoresis
  2. Agarose gel electrophoresis buffer
  3. DNA gel loading dye
  4. Role of EtBr in molecular genetics and cytogenetics

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