NaCl role in CTAB - DNA complex in DNA extraction

NaCl role in CTAB - DNA complex in DNA extraction

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I have a question about the role of NaCl in the DNA extraction process. So for NaCl concentrations under 0.5M, CTAB and DNA molecules can create complexes. In those concentrations, proteins and other hydrocarbons are still soluble in water except the DNA-CTAB complex. If we raise the concentration of NaCl then the complex of CTAB-DNA will be soluble to water too. Correct me if I'm wrong until now.

So I don't understand something. Why do we use NaCl?

Let's assume that we don't add NaCl. Will the CTAB create complex with DNA or not? As I read CTAB creates ionic bonds with DNA (phosphate groups). So in this situation (without NaCl) CTAB must be able to create complex with DNA too.

So why did we use NaCl? Just to keep proteins and other hydrocarbons soluble in the water or there is something else that NaCl helps in this complex creation?

CTAB forms insoluble complexes with nucleic acids and can be used to selectively precipitate them from solutions, see this reference:

When you add NaCl in a concentration between 0.4 - 0.7M, the nucleic acids stay in solution, while polysaccharides and other substances, which may interfere with the DNA preparation do not. See this paper: "Rapid isolation of high molecular weight plant DNA."

What Is the Purpose of Salt in DNA Extraction?

During the extraction of deoxyribonucleic acid, or DNA, salt compounds such as sodium acetate and ammonium acetate are typically added to aid in the removal of DNA-associated proteins. Another type of salt compound called sodium chloride, or NaCl, helps in solidifying and making DNA visible. When mixed in an alcohol solution, the sodium component of NaCl provides a protective barrier around the negatively-charged DNA phosphate ends, enabling them to move closer to be extracted out of the solution.

DNA extraction is the process of obtaining pure DNA from a sample, either from living or non-living cells, such as those found in viruses. This technique is commonly used in the medical field, where early detection of diseases and disorders significantly increases the survival rates of afflicted individuals.

The method initially requires the lysis of cells that contain the DNA to be extracted. The cells disintegrate by subjecting the sample to ultrasonic oscillations or by bead beating. The sample is added with salt, which is centrifuged in a solution of phenol-chloroform. The associated protein molecules are then drawn out. The DNA that is left after the removal of the proteins is mixed with an alcohol solution, typically cold isopropanol or ethanol. The solution is centrifuged, and DNA, which does not dissolve in alcohol, is precipitated and extracted. To increase DNA yield, the entire process must be performed in a cold environment.

What is DNA precipitation?

DNA precipitation is a process in which the DNA is precipitated or aggregated into the visible cotton thread like precipitates using alcohol and salt.

The DNA precipitation also removes the impurities from DNA.

“The precipitation is a process in which the reaction between solute and solvent creates insoluble aggregate called precipitate and the process is called precipitation.”

The DNA is hydrophilic in nature which dissolves in water but not in the alcohol.

The water has a partial positive charge and a partial negative charge. The positive charge of the water molecule interact with the negative charge of the DNA (the PO3-) and dissolve it. However, the interaction is not so strong.

The image shows the intermolecular interaction between water and DNA.

The salt and alcohol make the DNA more water hydrophobic, once we add the salt into the DNA, the negative charge of the DNA interact with the positive charge of the salt, instead of the positive charge of the water.

It might bind to the water but thanks to the alcohol, the “lower dielectric constant alcohol” protect the salt and DNA complex by shielding it against the water.

The DNA is visible like a cotton thread and aggregate as a precipitate due to this chemical reaction.

For a more in-depth explanation on the mechanism of the DNA precipitation please read our previous article: Role of alcohol in DNA extraction.

Furthermore, instead of the negative charge of the DNA, the water remains busy in interaction with the negative charge of the alcohol that also increase the precipitation efficiency and the overall yield of the DNA precipitate.

The entire mechanism of precipitation is based on the polarity of the solution and the dielectric constant of the solvent.


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Challenges during DNA Extraction and Downstream Applications

Food authentication requires DNA of high quality, as it is vital for polymerase chain reaction (PCR)-based analysis. The low quality of DNA extracted from food products following massive heat processing, obstructs the process of authentication (Demirhan, Ulca, & Senyuva, 2012 ). Selecting an appropriate method for DNA extraction takes into account certain factors, such as time, cost and toxicity of the chemicals employed (Chapela et al., 2007 ). For instance, a conventional method of extracting DNA from animal species generally requires the addition of phenol and chloroform (Lopera-Barrero et al., 2008 ), which pose greater risks of contaminating DNA and health hazards (Yue & Orban, 2001 ). The method is also time-consuming.

To ensure successful PCR amplification, the purity of the DNA extracted is of greater importance than the yield of DNA (Särkinen, Staats, Richardson, Cowan, & Bakker, 2012 ). Pinto et al. ( 2007 ) has described the presence of inhibitors or contaminants originating from foods, such as polysaccharides and humic acid, while Rijpens, Jannes, Asbroeck, Rossau, and Herman ( 1996 ) indicates protein in milk reduces the solubility of pelleted cells from which DNA is extracted. Wilson ( 1997 ) reported lipids and phenolic compounds that may contaminate the DNA. Furthermore, some chemical constituents employed during DNA extraction have been identified as PCR inhibitors. For instance, the chelating agent, ethylenediaminetetraacetic acid (EDTA), may form a complex with magnesium, which eventually affects the effective concentration of magnesium required for PCR. As a result, increased concentration of magnesium is necessary in order to support amplification (Khosravinia & Ramesha, 2007 ).

In addition, chemicals, such as phenol, causes polymerase to denature whereas NaOH also causes DNA and polymerase degradation. The usage of isopropanol and ethanol in DNA precipitation step affects the quality of DNA obtained (Bar, Kubista, & Tichopad, 2012 Hedman & Rådström, 2013 ). These inhibitors will hamper and interfere with downstream applications or they may cause complete inhibition of DNA polymerase activity in PCR (Pinto et al., 2007 ). Alternatively, PCR inhibitors may be eliminated through the spin column technique, where with the aid of chaotropic agents, DNA binds to silica membranes during extraction (Pinto, Forte, Conversano, & Tantillo, 2005 ).

Apart from inhibitors, increased temperatures and the duration of heat treatment can gradually degrade the size of DNA fragments (Şakalar, Abasiyanik, Bektik, & Tayyrov, 2012 ). Likewise, several factors, such as primer specificity, amplicon size and gene copy number, should be taken into account for successful amplification (Jagadeesan & Salem, 2017 ). Therefore, incorporating an endogenous control in downstream applications is important, as it may verify potential amplification variations due to certain factors. These factors, which may affect the outcome of the assay, include varying the amount and quality of DNA extracted from samples and DNA degradation (Soares, Amaral, Oliveira, & Mafra, 2013 ).

Materials and methods

Soybean seed sources

Five kinds of soybean seeds including non-biotech and biotech varieties were utilized in the present study. The non-biotech soybean seed materials, Zhoudou22, Zheng196, and Zhonghuang13, were purchased from Henan Academy of Agricultural Sciences. Two kinds of GM soybean seeds were obtained from Henan Sunshine Oils and Fats Group importing GM materials from America annually. Immunochromatography test strip assay had been performed revealing 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) positive for both the GM soybean seeds. Considering the GM soybean samples were geographically different, we named them Roundup Ready Soybean (RRS) 1 (RRS1) and Roundup Ready Soybean2 (RRS2) respectively.

Genomic DNA extraction protocols

All soya seeds were ground and homogenized in liquid nitrogen with a mixer mill, followed by filtrating through 80-mesh sieve. All ground samples were stored at −20°C prior to DNA extraction.

SDS method 1

One hundred milligram of Zhoudou22 was weighted and transferred to a 2-ml sterile centrifuge tube. One milliliter of SDS extraction buffer (20 g SDS/l, 150 mM NaCl, 100 mM Tris/HCl, 25 mM EDTA, pH 8.0) preheated at 65°C was added and mixed followed by adding 10 μl Proteinase K (10 mg/ml). Then, the reaction tube was incubated at 65°C for 1 h, with stirring every 10 min. After centrifuging the tube for 10 min at 12000×g, the supernatant was extracted twice with phenol/chloroform/isoamyl alcohol (P: C: I, 25: 24: 1, v/v/v) (first extraction) and chloroform/isoamyl alcohol (C: I, 24: 1, v/v) (second extraction), respectively. Then the upper aqueous phase was added with 0.1 volume potassium acetate solution (3 M, pH 5.5) and double volume of ethanol solution (95%, v/v, −20°C) (first precipitation), followed by gentle inversion and vortex for 10 min at 15000×g to pellet DNA. After washing the pellet with ethanol solution (70%, v/v, −20°C) twice and air drying for 5 min, the dried pellet was dissolved with 400 μl Tris/EDTA buffer (10 mM Tris, 1 mM EDTA). Ten milligram of RNase was added in the mixture and an incubation at 37°C for 30 min was performed to eliminate the remaining RNA. Another extraction with C:I (third extraction) was carried out to remove protein from DNA solution. Recovering the upper layer to a new sterile tube containing 2.5 vol of ethanol (second precipitation) would help precipitate DNA readily. After spinning tube at 15000×g for 10 min and washing DNA pellet twice, the dried DNA was redissolved in 200 μl sterile, deionized water.

CTAB method

The operation in this method was similar to that described in SDS method 1 except the lysis buffer and organic regents used to precipitate DNA. This method was based on CTAB extraction buffer (20 g CTAB/l, 1.4 M NaCl, 100 mM Tris/HCl, 20 mM EDTA, pH 8.0) and 0.6 vol of isopropanol was added to the upper aqueous phase to pellet DNA twice instead of ethanol solution used in SDS method 1.

DP305 and DNeasy plant mini kit method

Concerning two commercial kits, DP305 (TIANGEN, Beijing, China) and DNeasy Plant Mini kit (Qiagen, Hilden, Germany), DNA was extracted from Zhoudou22 according to the manufacturer’s instructions.

SDS method 2

The DNA extraction protocol was described in SDS method 1 except for the lysis buffer components. Concentrations of each component in SDS extraction buffer are detailed in Table 2.

Components composition of SDS lysis buffer (pH 8.0) .
Number . SDS (w/v) . NaCl (mM) . Tris/HCl (mM) . EDTA (mM) .
1 0.5% 250 100 25
2 2% 250 100 25
3 5% 250 100 25
4 2% 0 100 25
5 2% 150 100 25
6 2% 250 100 25
7 2% 500 100 25
8 2% 250 10 25
9 2% 250 50 25
10 2% 250 100 25
11 2% 250 200 25
12 2% 250 100 5
13 2% 250 100 25
14 2% 250 100 50
15 2% 250 100 100
Components composition of SDS lysis buffer (pH 8.0) .
Number . SDS (w/v) . NaCl (mM) . Tris/HCl (mM) . EDTA (mM) .
1 0.5% 250 100 25
2 2% 250 100 25
3 5% 250 100 25
4 2% 0 100 25
5 2% 150 100 25
6 2% 250 100 25
7 2% 500 100 25
8 2% 250 10 25
9 2% 250 50 25
10 2% 250 100 25
11 2% 250 200 25
12 2% 250 100 5
13 2% 250 100 25
14 2% 250 100 50
15 2% 250 100 100
Organic reagents used to extract and precipitate DNA .
Procedure . First extraction . Third extraction . First precipitation . Second precipitation .
A P: C: I C: I Ethanol Ethanol
B C: I C: I Ethanol Ethanol
C P: C: I / Ethanol Eethanol
D P: C: I C: I KAc + 95% Ethanol Ethanol
E P: C: I C: I Isopropanol Ethanol
F C: I C: I Ethanol Isopropanol
G C: I C: I Isopropanol Isopropanol
H C: I C: I Isopropanol Ethanol
Organic reagents used to extract and precipitate DNA .
Procedure . First extraction . Third extraction . First precipitation . Second precipitation .
A P: C: I C: I Ethanol Ethanol
B C: I C: I Ethanol Ethanol
C P: C: I / Ethanol Eethanol
D P: C: I C: I KAc + 95% Ethanol Ethanol
E P: C: I C: I Isopropanol Ethanol
F C: I C: I Ethanol Isopropanol
G C: I C: I Isopropanol Isopropanol
H C: I C: I Isopropanol Ethanol

SDS method 3

The DNA extraction protocol was described in SDS method 1 except for the organic solvent varieties. The use of organic solvents was shown in Table 2. The addition volume of isopropanol was 0.6 vol supernatant.

SDS method 4

It was the optimized DNA extraction method. Its protocol was shown in SDS method 1 except several modifications: SDS lysis buffer (2% SDS (w/v), 150 mM NaCl, 50 mM Tris/HCl, 50 mM EDTA, pH 8.0), first extraction reagent (C: I), third extraction reagent (C: I), first precipitation reagent (isopropanol), second precipitation reagent (ethanol).

Reagents used for DNA isolation and purification

We recently did an experiment on isolation and purification of DNA from bacterial cells and as hard as i try i really don't get what happened as a result of what reagent? I've looked online and in several texts but almost every source gives a different answer.

What we used (in order of use):
TE buffer
Proteinase K
CTAB/NaCl solution
Chloroform/isoamyl alcohol 24:1
Phenol/chloroform/isoamyl alcohol 25:24:1

SDS lyses cells
Proteinase K inactivates nucleases
NaCl neutralises charges on DNA
Phenol/chloroform/isoamyl separates into phases and removes 'gunk'
Isopropanol precipitates DNA
Ethanol removes salt

The rest i'm so confused about.

This is one of many different genomic DNA isolation protocols, although you leave us with a bit of guesswork as to the exact protocol! This type of method is also occasionally modified for use in plasmid DNA minipreps. SDS, an anionic surfactant (often found in many cleaning products like hair shampoo), does lyse the bacteria. In alkaline lysis plasmid DNA minipreps SDS is often used in conjunction with NaOH (e.g., 0.2N) following a lysozyme enzyme digestion, and then you would not typically have a proteinase K step. PK is more often used with genomic DNA isolation (without alkaline lysis) or even after a restriction enzyme digest. So proteinase K will inactivate nucleases (e.g., DNases/RNases) particularly in the presence of SDS, but it a broad-spectrum protease that also digests proteins to ensure complete lysis of the cells.

CTAB (Cetyltrimethylammonium bromide) is a cationic surfactant that further solubilizes the cells. In general DNA is ‘happy’ in NaCl - the Na+ ions do neutralize the negatively charged phosphates on DNA and facilitate DNA molecules coming together (the molecules are less hydrophilic). A high concentration of NaCl is required otherwise CTAB-nucleic acid precipitates can form - what you are trying to do here is remove all the junk (bacterial cell wall debris, denatured proteins, polysaccharides) complexed with CTAB and leave the bacterial DNA in solution. The chloroform/IAA extraction (with microfuging) leaves these complexes in a whitish interface (chloroform at the bottom, DNA at the top IAA helps stabilize the interface). The DNA is ‘cleaned up’ with phenol/chloroform/IAA is to remove any remaining complexes, and is precipitated from the supernatant with isopropanol (large, chromosomal DNA is easily precipitated with this non-polar alcohol, even without the addition of extra salt or using low temperatures). The pellet is washed with EtOH (usually but not always 70-75%) to remove any remaining isopropanol and residual salts.


Different species of Cinnamomum are rich in polysaccharide’s and secondary metabolites, which hinder the process of DNA extraction. High quality DNA is the pre-requisite for any molecular biology study. In this paper we report a modified method for high quality and quantity of DNA extraction from both lyophilized and non-lyophilized leaf samples. Protocol reported differs from the CTAB procedure by addition of higher concentration of salt and activated charcoal to remove the polysaccharides and polyphenols. Wide utility of the modified protocol was proved by DNA extraction from different woody species and 4 Cinnamomum species. Therefore, this protocol has also been validated in different species of plants containing high levels of polyphenols and polysaccharides. The extracted DNA showed perfect amplification when subjected to RAPD, restriction digestion and amplification with DNA barcoding primers. The DNA extraction protocol is reproducible and can be applied for any plant molecular biology study.

Crude extraction of DNA

Aim: To understand the procedure of DNA extraction using household materials.

Introduction: How does the crime scene investigator identify the suspect of murder case? How does the fisherman detect viral disease which causes outbreak in his ponds? How does your blood tell people who your parents are? How does the scientist discover the drought-resistance gene in plants? Those answers can be found in the field work of DNA technology.

  1. Extraction, to separate the DNA from the cell. This stage usually utilizes SDS/CTAB/Triton-X as active detergent and saturated NaCl
  2. Purification, to separate extracted DNA from any contaminant, cell debris, protein, and RNA. Scientists use phenol:chloroform:isoamylethanol (PCIA) for this purpose and
  3. Precipitation, to precipitate the DNA to form thin, transparent or white strands. Ice cold 96% ethanol can be used at this stage.
  1. Grind up 0.5 gram of sample in the sterile mortar and homogenize with the buffer solution,
  2. Filter the homogenate using filter paper and put in the sterile centrifuge.
  3. Mix the filtrate with 5 mL of SDS 20% and 5 mL of 5M NaCl using vortexer.
  4. Centrifuge the suspension to separate cellular components from cell debris.
  5. Remove the supernatant into new tube.

Add ice cold 96% ethanol carefully to the new tube through the side of the tube. There will be a white strands appear on the suspension surface if you conduct the DNA extraction correctly.

Comparison of Total DNA Extraction Methods for Microbial Community Form Polluted Soil ☆

DNA isolation represents the basic and probably the most important step in molecular biology for microbial strains, and even more, for microbial community analyses. Despite the development of molecular protocols for DNA microbial community isolation, there are still many drawbacks dependent of samples composition, and even the commercially available genomic isolation kits have significant limitations in recovering high genomic DNA amounts, especially from soil samples. Our study is aiming to compare and optimize a total microbial community DNA isolation protocol from polluted soil samples, estimating the amount and the purity of genomic DNA per g of soil, versus time requirements for each protocol, taking under consideration that our soil samples have a high content of humic acids. We checked several protocols for total DNA extraction, CTAB based, including a specific Kit for Soil DNA Isolation Kit NorgenBiotek. We estimated the time needed for each protocol, the amount of the DNA per gram of polluted soil, proteins and RNA contamination grade, by spectrophotometric analysis, but also the grade of PCR amplification inhibition. The most efficient method for our soil samples with high content of humic acid, suitable for further molecular analyses was the total DNA microbial community sample retrieved from Sagova et al. (2008) based protocol, with several adjustments. This protocol will be valuable for molecular analysis on microbial community profiling from environmental samples, especially from polluted soils.



  1. Zulkile

    Between us speaking, I would address for the help to a moderator.

  2. Amichai


  3. Secg

    It is true! The idea of ??a good, I agree with you.

  4. Daim

    I cannot participate in the discussion now - no free time. Osvobozhus - necessarily their observations.

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