AbstractDNA serves as the foundation for molecular biology, leading to the development of numerous molecular techniques. Often, these techniques necessitate the separation and visualization of specific DNA regions. Electrophoresis provides a solution for this requirement. However, the purification of DNA from agarose gels presents a significant challenge, both in terms of complexity and cost. Therefore, here we propose two main protocols that are both cost-effective and efficient based on silica columns or freezing follow by alcohol precipitation. In the case of silica column extraction, the gel was partially or completely dissolved, yielding DNA in most situations. In the case of extraction by freezing and precipitation with ethanol, DNA was obtained in only two out of three treatments. A successful bacterial transformation and PCR were achieved confirmed the suitability of the recovered DNA for further applications.
IntroductionDeoxyribonucleic Acid (DNA) is the cornerstone molecule in the field of molecular biology, serving as the repository for an organism’s genetic information. The comprehension and manipulation of this molecule are indispensable for the study and understanding of biological processes at a molecular level. Fundamental processes such as DNA replication, transcription, subsequent translation of this molecule, and mutations have led to the development of a plethora of techniques that employ DNA as their primary instrument. These techniques have revolutionized the domain of molecular biology and continue to be integral to scientific research.The Polymerase Chain Reaction (PCR) has emerged as a groundbreaking technique in the realm of Molecular Biology. Prior to its advent, serological tests were primarily employed for disease detection. However, these tests lacked the precision and convenience of early-stage disease detection, a gap that PCR has effectively bridged by identifying biomarkers1. PCR involves a series of repetitive cycles at varying temperatures, resulting in the production of millions of copies of a DNA molecule, referred to as a template2. This technique has proven to be instrumental in detecting diseases such as respiratory tract infections caused by bacteria3 or viruses4.Molecular cloning is another technique that has found extensive application. It is indispensable for the acquisition, isolation, and creation of gene libraries, as well as for expression of DNA fragments to yield recombinant proteins in various organisms, such as E. coli. This is achieved through the integration of vectors (i.e. plasmids), and utilization of a range of enzymes that facilitate the incorporation of fragments5. This technique has paved the way for the development and production of various systems, including the creation of personalized mRNA-based vaccines6, gene editing7 and the production of recombinant proteins for therapeutic applications8.In most instances, electrophoresis is the preferred method as it facilitates the separation and visualization of DNA fragments ranging from 100 bp to 25 Kb in agarose or polyacrylamide gels9,10. However, the extraction and purification of DNA from agarose gels can pose a significant challenge. Specialized kits designed for this purpose are often expensive in comparison with the use of reagents that are commonly found in a laboratory and are almost always available, rendering them inaccessible to all laboratories11. While alternative extraction methods have been documented, they frequently required equipment12,13 or reagents that are either difficult to procure, manipulate or not typically found in a standard laboratory inventory13. Consequently, the objective of the current work is to establish a method based on chaotropic salt and column adsorption or freezing followed by alcohol precipitation, for extracting DNA from agarose gels in a manner that is both cost-effective and straightforward with the utilization of reagents that are commonly employed in laboratory settings and can be procured at a reasonable cost thus contributing to the feasibility of this approach and that can be used for downstream applications.ResultsThe bands corresponding to the amplification of GFP contained in the PJet vector were visualized and excised from the gel. The objective was to extract the DNA present within these bands using five potential treatments. Among all the treatments, only the one involving TAE was used as negative control -only for column test treatment- failed to dissolve under the conditions analyzed. For the remaining treatments, the gel was either fully or partially dissolved (Table 1). Conversely, when the gel was frozen with slight mechanical disruption, all extraction steps could be performed in TAE or Trizol. Subsequent electrophoresis in 1.5% agarose was used to visualize the DNA recovered from each treatment. Only 6MC, Taf and TzF treatments exhibited defined bands without DNA degradation (Fig. 1A), whereas treatment 1 did not display any band, and treatment 2 showed a barely visible band indicating the presence of DNA.Table 1 Characteristics of the different treatments proposed for obtaining DNA from agarose gels.Full size tableFig. 1Agarose gel (1.5%) electrophoresis of PCR fragments using different purification methods. (A) Purification of PCR product (800 bp) incorporated into the PJet Blunt cloning vector. From left to right: DNA ladder, positive control, negative control. (B) Purification of PCR product (400 bp).Full size imageDNA fragments of different sizes often required purification. To verify that the DNA extraction from 6MC, TAF and TzF could be used to extract shorter or larger DNA fragments, such as plasmids, two additional DNA samples were isolated from electrophoresis on a 1% agarose gel. One sample corresponded to a 2958 bp (bp) plasmid and the other was a 600 bp PCR fragment. In both cases, it was possible to extract DNA from the agarose gels regardless of the method used (Figs. 1B and 2B). However, for the plasmid, the amount of DNA recovered was lower than expected in the case of the silica column treatment. This could be due to a higher affinity of the DNA for the silica matrix in the column, making it difficult to extract.Fig. 2Downstream applications for DNA agarose extraction. (A) PCR amplification from DNA plasmid recovered from 6MC and TaF. (B) Cloning and restriction from Plasmid recovered from 6MC and TaF, The first three columns after DNA ladder shown the plasmid from transfected E. coli cells, The last three columns shown the restriction from plasmids recovered from 6MC and TaF.Full size imageTo confirm the suitability of the recovered DNA for further applications, polymerase chain reaction (PCR) was performed on the DNA recovered from 6MC, TAF or TzF of the PJet-GFP plasmid using the PJet-specific primers. Of these, only 6MC and TAF could be amplified during the PCR reaction (Fig. 2A). Extractions of the Pjet-GFP plasmid on a 1% fatty acid gel were also performed with treatments 6MC, TAF or TzF for transformation into chemically competent E. coli TOP10 cells with this plasmid or for digestion with the restriction enzyme MspI. The transformation was achieved in treatments 6MC and TAF (Fig. 2B).DiscussionThe extraction and purification of DNA is a fundamental process in all aspects of molecular biology, ranging from disease diagnosis, such as cancer, to cloning procedures, and the identification of GMOs or infectious agents. To accomplish this, it is often necessary to visualize the DNA of interest using techniques such as agarose or polyacrylamide gel electrophoresis10, Once the relevant DNA bands have been identified, they are directly purified from the gel14. This extraction is typically performed using commercial kits. However, access to these products is not always possible. As a result, several techniques have been proposed for extracting DNA from agarose gels, including the freeze-squeeze technique15, phase separation and quaternary ammonium16, organic extraction17, and centrifugation18. In the current study, DNA was successfully extracted from agarose gels using two different, cost-effective, and straightforward techniques. The first technique is based on the use of chaotropic salts, which facilitate the dissolution of agarose and subsequent binding to a silica matrix, followed by elution from the matrix. The second technique involves disrupting the gel first by freezing and then mechanically, followed by alcohol precipitation. In both cases, the samples are centrifuged to separate and precipitate the components.Gel dissolution is achieved in the first technique by solubilizing the agarose gel at moderate temperatures in a solution of chaotropic salt solution such as KI19 or the guanidine isothiocyanate into the TRIzol20. The chaotropic disrupts hydrogen bonds and disarranges water molecules in an aqueous environment, which disrupts the secondary structure of polymers such as agarose gels21 and allows DNA to bind to the silica matrix of the column, resulting in adsorption of DNA to the silica matrix22, allowing separation of nucleic acids from contaminants. The lack of melting of agarose with TAE at 50 °C prevents its loading onto the silica matrix. This is partly because the agarose present in the gel is soluble in water up to 85 °C23, which could degrade the DNA. Furthermore, the pH of the TAE buffer 8.2–8.4 is not suitable for DNA binding to the silica matrix, as an acidic pH is required22. Therefore, this treatment was chosen as a negative control for DNA extraction using silica columns.The second technique proposed for DNA recovery is to the agarose gel by first freezing it to alter the structure of the agarose by destroying the polymer caused by the ice crystals accompanied by mechanical disruption to aid this process allowing elution of the DNA from the agarose gel24.An inverse relationship exists between the concentration of the agarose gel and the quantity of recoverable DNA15. In the case of using chaotropic salts to dissolve the gel, it is recommended that gels with a concentration of 1% or less be used. For the case of the freezing method, agarose gels with concentrations of 1–2% are typically utilized11, accordingly, our research was conducted using 1% agarose gels. This concentration facilitated the dissolution of the gel, resulting in an agarose solution that did not re-solidify23. Contrary to methods involving phase separation and the use of quaternary ammonium cations or organic extraction, these compounds can interfere with the subsequent use of the extracted DNA in other applications, such as Polymerase Chain Reaction25, the failure of the PCR amplification in treatment 2 may have been due to the presence of phenol in TRIzol, which has been demonstrated to inhibit Taq Polymerase26. One potential solution to this issue is the purification of DNA.Additionally, our method avoids the need to eliminate quaternary ammonium cations during extraction, and it does not require any additional steps or the preparation of extra solutions beyond those typically used in the laboratory, this simplicity could be advantageous in resource-limited settings. It is important to highlight that DNA fragments of varying sizes were successfully obtained. These fragments were visualized using ethidium bromide, a process that may have implications for the potential applications of the extracted DNA, such as PCR25. In both methods employed, the complete removal of ethidium bromide cannot be assured. To address this, the extracted DNA can be purified using a phenol-chloroform method27. Despite the uncertainty surrounding the complete removal of ethidium bromide, the extracted DNA could still be utilized for PCR, restriction analysis or cloning However, in this last section it is necessary to be careful when choosing the type of column to be used, since the number of silica layers present in the column can modify the yield about the amount of plasmid DNA obtained, since due to its size and secondary structure it has a greater affinity to the column, affecting the elution phase and making it more difficult. This suggests that both methods employed are potentially viable for subsequent applications.It is important to note that the proposed methods may have inherent limitations. In the case of extraction using chaotropic salts and columns, KI may not always be available. However, it can be replaced by NaClO₄28 or NaI29. Another potential disadvantage is that the selected columns may not be of the highest quality, and the repeatability of the test may vary between batches or brands. The method of freezing and subsequent precipitation with ethanol has the disadvantage of requiring up to a day’s time, or of being inconvenient for processing multiple samples simultaneously. However, both methods are straightforward to implement without the need for additional equipment and may be susceptible to improvement.MethodsA volume of 1µL the Pjet-GFP plasmid extracted from E. Coli TOP10 cells was used as template, the primers used were specific for PJET (Fw 5’-CGACTCACTATAGGGAGGGAGAGCGGC-3’), Rv (5’-AAGAACATCGATTTTCCATGGCAG-3’), they flanked the GFP sequence in the vector, PCR buffer, deoxynucleotide triphosphates and Taq polymerase (QIAGEN, Germany) were added to achieve a final volume of 20 µL. The amplification was executed using an Applied Biosystem 9700 thermal cycler under specific conditions: an initial denaturation at 94 °C for 10 min, followed by 25 cycles that included a 94 °C denaturation, alignment at 58 °C, and extension at 72 °C. This was concluded with a final extension at 72 °C for 10 min. The samples were then stored at 4 °C for future use.The electrophoresis of PCR products was conducted in a 1% agarose gel (Sigma-Aldrich, USA), utilizing ethidium bromide to visualize the bands via a transilluminator (ANT Technology). The bands were carefully excised, with an aim to retain minimal agarose. The DNA fragments embedded in the agarose underwent two main procedures: column extraction and mechanical disruption. For column extraction the gel was dissolved by heating at 50 °C for 10 min, followed by extraction on generic silica columns employing tree treatments: 3 M KI (3MC), 6 M KI (6MC), or Trizol (TzC) and TAE for column or H2O dd as negative control (C-) for mechanical disruption and the NucleoSpin Gel and PCR Clean-up kit (MACHENERY-NAGEL, Germany) as positive control (C+). Followed by three washes with wash buffer (NaCl 50 mM, Tris HCl 10mM, EtOH 70%). The elution was performed in 35 µL of Buffer TE,For mechanical disruption, the gel was frozen at -20 °C for 24 h, followed by mechanical fragmentation using a polystyrene tip and heating at 50 °C for 10 min. This process comprised two treatments: gel plus TAE (TaF), or gel plus Trizol (TzF). After heating, the DNA extraction was carried out by centrifugation for 5 min at 12,000 x g, supernatants were transferred into a clean 1.5 mL centrifuge tube and 500 µL of absolute EtOH and 50 µL of CH3COONa were added, followed by cooling at -20 °C for 30 min, centrifugation, washing with 70% EtOH, and elution in 35 µL of buffer TE. For more detailed information regarding the protocol, please refer to Appendix 1. To validate the feasibility of the methods for small DNA fragments or larger DNA lengths, the other two samples (600 bp PCR product or Pjet-GFP plasmid 2958 bp) were extracted and visualized under the same conditions.To verify the quality of the extracted DNA, the samples were subjected to electrophoresis in a 1.5% agarose gel, utilizing ethidium bromide for visualization under a transilluminator. To ascertain the viability of DNA extractions for subsequent applications, the Pjet-GFP plasmid was extracted on a 1% agarose gel and a PCR was conducted under the previously established conditions. Additionally, a restriction analysis was performed using the enzyme MspI (Promega, USA), which cut restriction sites present in the plasmid, or clone and transform E. coli TOP10. Subsequently, the results were validated through electrophoresis in a 1.5% agarose gel.
Data availability
All data supporting the findings of this study are available within the paper and its Supplementary Information. primer sequences are provided in methods, steps of protocol are detailed in supplementary information. A well the data that support the findings of this study are available from the corresponding author.
ReferencesNazir, S. Medical diagnostic value of digital PCR (dPCR): A systematic review. Biomed. Eng. Adv. 6, 100092 (2023).MATH
Google Scholar
Son, J. H. et al. Ultrafast photonic PCR. Light Sci. Appl. 4, (2015).Asencio Egea, M. Á., Gaitán Pitera, J., Méndez González, J. C. & Huertas Vaquero, M. Utility of multiplex PCR syndromic panel for respiratory infections in the diagnosis of acute bacterial meningitis. Enfermedades Infecc Y Microbiol. Clin. (English ed.). 39, 481–482 (2021).A, M. Y., Filiztekin, E. & Ozkaya, K. G. COVID-19 diagnosis — A review of current methods. Biosens. Bioelectron. 172, 1–20 (2021).MATH
Google Scholar
Bertero, A., Brown, S. & Vallier, L. Methods of Cloning. Basic Science Methods for Clinical Researchers (Elsevier Inc., 2017). https://doi.org/10.1016/B978-0-12-803077-6.00002-3.Yarchoan, M. et al. Personalized neoantigen vaccine and pembrolizumab in advanced hepatocellular carcinoma: A phase 1/2 trial. Nat. Med. 30, (2024).Singh, A. et al. A high efficiency precision genome editing method with CRISPR in iPSCs. Sci. Rep. 14, 9933 (2024).ADS
CAS
PubMed
PubMed Central
MATH
Google Scholar
Zhang, R. X. et al. Intravitreal injection of fibrillin 2 (Fbn2) recombinant protein for therapy of retinopathy in a retina-specific Fbn2 knock-down mouse model. Sci. Rep. 13, 1–13 (2023).
Google Scholar
Priyashantha, A. K. H. & Umashankar, S. Advances in Biotechnology and Bioscience. in Advances in Baiotechnology and Bioscience (ed Kumar, D. S.) 45–60 (AkiNik, 2021). https://doi.org/10.22271/ed.book.1405.Lee, P. Y., Costumbrado, J., Hsu, C. Y. & Kim, Y. H. Agarose gel electrophoresis for the separation of DNA fragments. J. Vis. Exp. 1–5. https://doi.org/10.3791/3923 (2012).Gao, X. et al. A reassessment of several erstwhile methods for isolating DNA fragments from agarose gels. 3 Biotech. 11, 1–7 (2021).MATH
Google Scholar
Smith, H. O. [46] Recovery of DNA from gels. Methods Enzymol. 65, 371–380 (1980).CAS
PubMed
MATH
Google Scholar
Yu, S. et al. Rapid recovery of DNA from agarose gel slices by coupling electroelution with monolithic SPE. Electrophoresis 30, 2110–2116 (2009).CAS
PubMed
MATH
Google Scholar
But, G. W. C. et al. Comparison of DNA extraction methods on CITES-listed timber species and application in species authentication of commercial products using DNA barcoding. Sci. Rep. 13, 1–15 (2023).MATH
Google Scholar
Tautz, D. & Renz, M. An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal. Biochem. 132, 14–19 (1983).CAS
PubMed
MATH
Google Scholar
Langridge, J., Langridge, P. & Bergquist, P. L. Extraction of nucleic acids from agarose gels. Anal. Biochem. 103, 264–271 (1980).CAS
PubMed
Google Scholar
Green, M. & Sambrook, R. (ed, J.) Recovery of DNA from low-melting-temperature agarose gels: Organic extraction. Cold Spring Harb Protoc. 2020 85–89 (2020).MATH
Google Scholar
Wang, Z. & Rossman, T. G. Isolation of DNA fragments from agarose gel by centrifugation. Nucleic Acids Res. 22, 2862–2863 (1994).CAS
PubMed
PubMed Central
MATH
Google Scholar
Shao, Q., Wang, J. & Zhu, W. On the influence of the mixture of denaturants on protein structure stability: A molecular dynamics study. Chem. Phys. 441, 38–46 (2014).ADS
CAS
MATH
Google Scholar
Sharma, R. et al. Academic Press,. DNA, RNA isolation, primer designing, sequence submission, and phylogenetic analysis. in Basic Biotechniques for Bioprocess and Bioentrepreneurship (ed. A. K. Bhatt, R. K. Bhatia, and T. C. B.) 197–206. https://doi.org/10.1016/B978-0-12-816109-8.00012-X (2023).Löwenberg, C., Julich-Gruner, K. K., Neffe, A. T., Behl, M. & Lendlein, A. Salt-Induced shape-memory effect in gelatin-based hydrogels. Biomacromolecules 21, 2024–2031 (2020).PubMed
Google Scholar
Katevatis, C., Fan, A. & Klapperich, C. M. Low concentration DNA extraction and recovery using a silica solid phase. PLoS One 12, 1–14 (2017).
Google Scholar
Ghebremedhin, M., Seiffert, S. & Vilgis, T. A. Physics of agarose fluid gels: Rheological properties and microstructure. Curr. Res. Food Sci. 4, 436–448 (2021).CAS
PubMed
PubMed Central
MATH
Google Scholar
Sasagawa, N. A freeze-and-thaw method to reuse agarose gels for DNA electrophoresis. Biosci. Trends 12, 627–629 (2018).CAS
PubMed
MATH
Google Scholar
Nath, K., Sarosy, J. W., Hahn, J. & Di Como, C. J. Effects of ethidium bromide and SYBR(®) Green I on different polymerase chain reaction systems. J. Biochem. Biophys. Methods 42, 15–29 (2000).CAS
PubMed
Google Scholar
Unger, C., Lokmer, N., Lehmann, D. & Axmann, I. M. Detection of phenol contamination in RNA samples and its impact on qRT-PCR results. Anal. Biochem. 571, 49–52 (2019).CAS
PubMed
Google Scholar
Lee, H. J., Lee, Y. L., Ji, J. J. & Lim, H. M. The effects of ethidium bromide and Mg++ ion on strand exchange in the Hin-mediated inversion reaction. Mol. Cells 16, 377–384 (2003).CAS
PubMed
MATH
Google Scholar
Chen, C. W. & Thomas, C. A. Recovery of DNA segments from agarose gels. Anal. Biochem. 101, 339–341 (1980).CAS
PubMed
MATH
Google Scholar
Chung, Y. B. & Na, W. J. Electroelution into an agarose-plugged Salt Channel. Mol. Cells 5, 243–247 (1995).CAS
Google Scholar
Download referencesAuthor informationAuthor notesJesús Enrique Sánchez-Flores, Antonio Sandoval-Cabrera, Patricia Alarcón-Valdés and Jonnathan Guadalupe Santillán-Benítez have contributed equally to this work.Authors and AffiliationsFaculty of Chemistry, Autonomous University of The State of Mexico, Toluca, 50120, State of Mexico, MéxicoJesús Enrique Sánchez-Flores & Jonnathan Guadalupe Santillán-BenítezHemato-Oncology High Specialty Laboratory, Childrens Hospital, Maternal and Child Institute of the State of Mexico, Toluca, 50120, Mexico State, MéxicoAntonio Sandoval-CabreraMedical Faculty, Autonomous University of Mexico State, Toluca, 50180, Mexico State, MéxicoAntonio Sandoval-CabreraHealth Research and Studies Institute, University of Ixtlahuaca CUI, Ixtlahuaca, 50740, Mexico State, MéxicoPatricia Alarcón-ValdésAuthorsJesús Enrique Sánchez-FloresView author publicationsYou can also search for this author inPubMed Google ScholarAntonio Sandoval-CabreraView author publicationsYou can also search for this author inPubMed Google ScholarPatricia Alarcón-ValdésView author publicationsYou can also search for this author inPubMed Google ScholarJonnathan Guadalupe Santillán-BenítezView author publicationsYou can also search for this author inPubMed Google ScholarContributionsJ. S-F. conception and design of the work, conducted the experiments analysis of data, revised it acquisition, interpretation of data, wrote the main manuscript text. A. S-C. design of the work, interpretation of data, analyzed the results, have substantively revised it. P.A-V. analyzed the results, have substantively revised it. J. S-B design of the work, interpretation of data, analyzed the results, have substantively revised it, approve the final version.Corresponding authorCorrespondence to
Jonnathan Guadalupe Santillán-Benítez.Ethics declarations
Competing interests
The authors declare no competing interests.
Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Electronic supplementary materialBelow is the link to the electronic supplementary material.Supplementary Material 1Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Reprints and permissionsAbout this articleCite this articleSánchez-Flores, J.E., Sandoval-Cabrera, A., Alarcón-Valdés, P. et al. An affordable and simple method for DNA extraction from agarose suitable for downstream applications.
Sci Rep 15, 10414 (2025). https://doi.org/10.1038/s41598-025-87572-wDownload citationReceived: 21 May 2024Accepted: 20 January 2025Published: 26 March 2025DOI: https://doi.org/10.1038/s41598-025-87572-wShare this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard
Provided by the Springer Nature SharedIt content-sharing initiative
KeywordsElectrophoresisDNARecovery