Name: Whole-Genome Deoxyribonucleic Acid Extraction from Mycobacterium Species via the Cetyltrimethylammonium Bromide Technique (Video) | JoVE Journal
Uploaded: 2025-12-12T12:40:00.000+00:00
Duration: 6 min 46 s
Description: Learn the CTAB method for extracting high-quality DNA from Mycobacterium species with robust cell walls. This protocol yields pure genomic DNA suitable for sequencing and molecular studies.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The authors express their sincere gratitude to the TB genomics team: Nabila Ismail, Melanie Grobbelaar, Emylin Costa, and Rob M Warren, for their contributions to the development and optimization of this protocol, as well as for their invaluable support throughout this study. This work and the people involved were supported by grants and funding from the South African Medical Research Council (SAMRC), National Research Foundation (NRF), and Stellenbosch University (SU). The funders had no role in the study design, data collection, analysis, or manuscript preparation.

The representative results were generated with the approval of the REC: BES ethics committee, Stellenbosch University (BES-2024-22504). The reagents and the equipment used are listed in the Table of Materials.
1. Preparation of mycobacterial culture suspension
NOTE: Depending on the species being examined, this step must be conducted in either a Biosafety level 2 (BSL-2) or a Biosafety level 3 (BSL-3) laboratory. The incubation time and conditions of the culture also depend on the mycobacterial species&#39; growth requirements due to their distinct growth rates and physiological characteristics.
<ol>
	<li>Prepare 5 mL or 8 mL of bacterial culture in either Middlebrook 7H9 liquid media or MGIT media, respectively, supplemented with OADC and incubate at 37 &#176;C for the required period to allow bacterial growth. Slow-growing mycobacteria require 10-14 days, while fast-growing mycobacteria require approximately 2-5 days. 
	NOTE: A suspension of mycobacterial cells can be prepared by scraping a loopful of colonies into sterile double-distilled water if the starting culture is from solid agar plates (e.g., Middlebrook 7H10 agar).</li>
	<li>Pour the liquid culture into 15 mL tubes.</li>
	<li>Heat-kill the culture at 80 &#176;C for 1 h in an incubator.</li>
	<li>In the case of Mycobacterium tuberculosis or other pathogenic species that require BSL-3, transfer the heat-killed cultures to the BSL-2 laboratory.</li>
</ol>
2. Cell wall digestion and DNA extraction
NOTE: Once the culture is heat-inactivated, the following steps can be safely carried out in a Biosafety level 2 (BSL-2) laboratory. If the goal is extraction for long-read sequencing or alternative applications requiring high molecular weight DNA, be sure to utilize wide-bore pipette tips, and at vortex steps, replace vortex with inversion or pipette mixing.
<ol>
	<li>Centrifuge the 15 mL tube at 3220 x g for 15-30 min at room temperature (RT). Discard the clear supernatant (ensure all the media is removed).</li>
	<li>Thoroughly resuspend the culture pellet in 300 &#181;L of Tris-EDTA (TE) buffer.</li>
	<li>Transfer the resuspended pellet into a 2 mL tube.</li>
	<li>Add 100 &#181;L of lysozyme (10 mg/mL) and mix by pipetting up and down 5 times, then gently tap the tube to ensure uniform dispersion.</li>
	<li>Incubate the mixture at 37 &#176;C on a rotary incubator overnight (~16 h; lower incubation time might lower the lysis efficiency and thus lead to low DNA yield).</li>
	<li>The following day, prepare a mixture of 5 &#181;L of proteinase K (10 mg/mL) and 70 &#181;L of 10% SDS. For example, if there are five samples, prepare 350 &#181;L of 10% SDS and 25 &#181;L of Proteinase K. Mix by inverting the tube to avoid foam formation.</li>
	<li>Add 75 &#181;L of the mixture to each sample.</li>
	<li>Mix by tapping and incubate at 65 &#176;C for 10 min in an incubator or heating block, with intermittent mixing by inversion.</li>
	<li>Add 100 &#181;L of 5 M NaCl.</li>
	<li>Add 100 &#181;L of preheated CTAB/NaCl solution at 65 &#176;C. 
	NOTE: Heat the CTAB/NaCl solution to 65 &#176;C before ensuring pipetting accuracy, considering the viscosity of the substance at RT.</li>
	<li>Mix by tapping until the solution becomes milky.</li>
	<li>Incubate at at 65 &#176;C for 10 min in an incubator or heating block, with intermittent mixing by inversion.</li>
	<li>Add an equal volume (~ 675 &#181;L) of chloroform/isoamyl alcohol solution (24:1) to each sample and mix by tapping. 
	NOTE: Perform this step in a fume hood as chloroform is toxic if inhaled and requires cautious handling.</li>
	<li>Centrifuge the samples at 12000 x g for 10 min at room temperature.</li>
	<li>Carefully aspirate 550-600 &#181;L of the aqueous (top) phase into sterile 1.5 mL tubes and label them appropriately. 
	NOTE: Be careful not to disturb the other layers. If any layers are inadvertently disrupted, centrifuge the tube again.</li>
</ol>
3. DNA precipitation and elution
<ol>
	<li>Add an equal volume (550-600 &#181;L) of ice-cold isopropanol and mix by inverting the tubes. 
	NOTE: If the DNA concentration is high, white precipitation may form during mixing.</li>
	<li>Incubate the tubes at -20 &#176;C for 30 min to 1 h. 
	NOTE: Longer incubation times can enhance the DNA precipitation18 but may also lead to the coprecipitation of salts. Thus, overnight incubation can be performed if a low DNA concentration is expected.</li>
	<li>Centrifuge at the highest speed (~21130 x g) for 30 min at room temperature to pellet the insoluble DNA. 
	NOTE: Ensure the front side of the 1.5 tubes faces inward in the centrifuge rotor. At this step, the DNA precipitates at the bottom and along the back of the tubes. This is important to avoid disturbing the possibly invisible pellet in the next step. A white precipitate or pellet may be visible if the DNA concentration is high. The absence of a pellet does not indicate a failed extraction; proceed with the remaining steps.</li>
	<li>Aspirate the supernatant from the front side of the tube without disturbing the DNA pellet or decanting it quickly while ensuring the pellet remains undisturbed. 
	NOTE: It is better to aspirate the supernatant if the starting culture is small and a low DNA concentration is expected.</li>
	<li>Add 1000 &#181;L of ice-cold 75% ethanol.</li>
	<li>Mix by inverting the tubes several times.</li>
	<li>Centrifuge at 12000 &#215; g for 30 min (in the same orientation as in step 3).</li>
	<li>Aspirate or decant all ethanol without disturbing the pellet. 
	NOTE: Steps 5-8 may be repeated if high salt coprecipitation is anticipated.</li>
	<li>Allow tubes to air dry at RT overnight or for at least 30 min.</li>
	<li>Add 25-50 &#181;L of TE buffer (pH 8) to each tube to resuspend the DNA. 
	NOTE: The DNA can be resuspended in Tris (10 mM, pH 8) buffer or Nuclease-free water if downstream applications are sensitive to EDTA.</li>
	<li>Allow the DNA pellet to resuspend overnight at 4 &#176;C.</li>
	<li>Store at 4 &#176;C for 2-3 days before performing a quality control assessment.</li>
	<li>Store at -80 &#176;C for long-term.</li>
</ol>
4. DNA quantification and quality control
NOTE: DNA quality control is generally performed using a microvolume spectrophotometer and gel electrophoresis. A fluorescence-based quantification system can also be used to quantify extracted DNA.
<ol>
	<li>Spectrophotometer method
	<ol>
		<li>Measure the DNA concentration and determine the quality following the manufacturer&#39;s instructions. 
		NOTE: A DNA sample ideally has a 260/280 ratio between 1.7 and 2.0. A value of 1.8 is usually considered pure for DNA. Values below this range indicate contamination by residual protein, guanidine, or other reagents that absorb strongly at or near 280 nm. A 260/230 ratio between 2.0-2.2 is ideal. If the ratio is lower than expected, this may indicate the presence of contaminants that absorb at 230 nm, such as EDTA, phenol, and carbohydrates. Check the absorbance graphs obtained for the sample as well.</li>
	</ol>
	</li>
	<li>Gel electrophoresis
	<ol>
		<li>Perform agarose gel electrophoresis19 to assess DNA integrity and determine whether fragmentation or degradation has occurred.</li>
		<li>To visualize DNA under the UV gel documentation system, use a 1.0% agarose gel containing SYBR Safe dye. Run the gel at 80 V for 45 min. 
		NOTE: The aim is to have clear, distinct, high-molecular-weight bands above the highest band of the DNA ladder. Use fresh 1&#215; TAE buffer each time.</li>
	</ol>
	</li>
</ol>

<ol><li>FBouso, J. M., Planet, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complete+non-tuberculous+mycobacteria+whole+genomes+using+an+optimized+DNA+extraction+protocol+for+long-read+sequencing%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">Complete non-tuberculous mycobacteria whole genomes using an optimized DNA extraction protocol for long-read sequencing.</a> BMC Genomics. 20 (1), 793(2019).</li>
<li>Daffé, M., Marrakchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unraveling+the+structure+of+the+mycobacterial+envelope%5BTitle%5D)+AND+%22Microbiol+Spectr%22%5BJournal%5D)">Unraveling the structure of the mycobacterial envelope.</a> Microbiol Spectr. 7 (4), 7.4.1(2019).</li>
<li>Wayne, L. G., Sohaskey, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nonreplicating+persistence+of+Mycobacterium+tuberculosis%5BTitle%5D)+AND+%22Annu+Rev+Microbiol%22%5BJournal%5D)">Nonreplicating persistence of Mycobacterium tuberculosis.</a> Annu Rev Microbiol. 55 (1), 139-163 (2001).</li>
<li>Mohammadi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimal+DNA+isolation+method+for+detection+of+non-tuberculous+mycobacteria+by+polymerase+chain+reaction%5BTitle%5D)+AND+%22Adv+Biomed+Res%22%5BJournal%5D)">Optimal DNA isolation method for detection of non-tuberculous mycobacteria by polymerase chain reaction.</a> Adv Biomed Res. 6 (1), 133(2017).</li>
<li>Van Soolingen, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Occurrence+and+stability+of+insertion+sequences+in+Mycobacterium+tuberculosis+complex+strains%3A+Evaluation+of+an+insertion+sequence-dependent+DNA+polymorphism+as+a+tool+in+the+epidemiology+of+tuberculosis%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: Evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis.</a> J Clin Microbiol. 29 (11), 2578-2586 (1991).</li>
<li>Wilson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation+of+genomic+DNA+from+bacteria%5BTitle%5D)+AND+%22Curr+Protoc+Mol+Biol%22%5BJournal%5D)">Preparation of genomic DNA from bacteria.</a> Curr Protoc Mol Biol. 56 (1), 2.4.1-2.4.5 (2001).</li>
<li>Van Embden, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strain+identification+of+Mycobacterium+tuberculosis+by+DNA+fingerprinting%3A+Recommendations+for+a+standardized+methodology%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: Recommendations for a standardized methodology.</a> J Clin Microbiol. 31 (2), 406-409 (1993).</li>
<li>Wang, W. -F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genomic+analysis+of+Mycobacterium+tuberculosis+isolates+and+construction+of+a+Beijing+lineage+reference+genome%5BTitle%5D)+AND+%22Genome+Biol+Evol%22%5BJournal%5D)">Genomic analysis of Mycobacterium tuberculosis isolates and construction of a Beijing lineage reference genome.</a> Genome Biol Evol. 12 (2), 3890-3905 (2020).</li>
<li>Epperson, L. E., Strong, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+scalable%2C+efficient%2C+and+safe+method+to+prepare+high+quality+DNA+from+mycobacteria+and+other+challenging+cells%5BTitle%5D)+AND+%22J+Clin+Tuberc+Other+Mycobact+Dis%22%5BJournal%5D)">A scalable, efficient, and safe method to prepare high quality DNA from mycobacteria and other challenging cells.</a> J Clin Tuberc Other Mycobact Dis. 19, 100150(2020).</li>
<li>Warren, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Safe+Mycobacterium+tuberculosis+DNA+extraction+method+that+does+not+compromise+integrity%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Safe Mycobacterium tuberculosis DNA extraction method that does not compromise integrity.</a> J Clin Microbiol. 44 (1), 254-256 (2006).</li>
<li>Amaro, A., Duarte, E., Amado, A., Ferronha, H., Botelho, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+three+DNA+extraction+methods+for+Mycobacterium+bovis%2C+Mycobacterium+tuberculosis+and+Mycobacterium+avium+subsp.+avium%5BTitle%5D)+AND+%22Lett+Appl+Microbiol%22%5BJournal%5D)">Comparison of three DNA extraction methods for Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium subsp. avium.</a> Lett Appl Microbiol. 47 (1), 8-11 (2008).</li>
<li>Arslan, N., Demiray-Gurbuz, E., Ozkutuk, N., Esen, N., Özkütük, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+four+different+DNA+isolation+methods+from+MGIT+culture+for+long-read+whole+genome+sequencing+of+Mycobacterium+tuberculosis%5BTitle%5D)+AND+%22Jundishapur+J+Microbiol%22%5BJournal%5D)">Comparison of four different DNA isolation methods from MGIT culture for long-read whole genome sequencing of Mycobacterium tuberculosis.</a> Jundishapur J Microbiol. 17 (9), e148070(2024).</li>
<li>Opperman, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mycobacterium+avium+DNA+extraction%3A+Implications+for+NTM+identification+and+amplicon+sequencing%5BTitle%5D)+AND+%22South+Afr+J+Infect+Dis%22%5BJournal%5D)">Mycobacterium avium DNA extraction: Implications for NTM identification and amplicon sequencing.</a> South Afr J Infect Dis. 40 (1), 5(2025).</li>
<li>Froenicke, L. Illumina and Aviti sample and library requirements. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aFroenicke%2C+L+%22DNA+Technol+Core%22">DNA Technol Core</a>. , https://dnatech.ucdavis.edu/illumina-aviti-sample-and-library-requirements (2015).</li>
<li>Froenicke, L. PacBio Revio and Sequel II library prep & sequencing. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aFroenicke%2C+L+%22DNA+Technol+Core%22">DNA Technol Core</a>. , https://dnatech.ucdavis.edu/pacbio-revio-and-sequel-ii-library-prep-sequencing (2016).</li>
<li>Dippenaar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Droplet-based+whole+genome+amplification+for+sequencing+minute+amounts+of+purified+Mycobacterium+tuberculosis+DNA%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Droplet-based whole genome amplification for sequencing minute amounts of purified Mycobacterium tuberculosis DNA.</a> Sci Rep. 14, 9931(2024).</li>
<li>Doig, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+efficacy+of+the+heat-killing+of+Mycobacterium+tuberculosis%5BTitle%5D)+AND+%22J+Clin+Pathol%22%5BJournal%5D)">The efficacy of the heat-killing of Mycobacterium tuberculosis.</a> J Clin Pathol. 55 (10), 778-779 (2002).</li>
<li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+systematic+investigation+of+key+factors+of+nucleic+acid+precipitation+toward+optimized+DNA%2FRNA+isolation%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">A systematic investigation of key factors of nucleic acid precipitation toward optimized DNA/RNA isolation.</a> Biotechniques. 68 (4), 191-199 (2020).</li>
<li>Green, M. R., Sambrook, J., Sambrook, J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGreen%2C+MR+%22Molecular+cloning%3A+A+laboratory+manual%22">Molecular cloning: A laboratory manual</a>. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor. N.Y. (2012).</li>
<li>Van Soolingen, D., Hermans, P. W., De Haas, P. E., Van Embden, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insertion+element+IS1081-associated+restriction+fragment+length+polymorphisms+in+Mycobacterium+tuberculosis+complex+species%3A+A+reliable+tool+for+recognizing+Mycobacterium+bovis+BCG%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Insertion element IS1081-associated restriction fragment length polymorphisms in Mycobacterium tuberculosis complex species: A reliable tool for recognizing Mycobacterium bovis BCG.</a> J Clin Microbiol. 30 (7), 1772-1777 (1992).</li>
<li>Doig, C., Seagar, A. L., Watt, B., Forbes, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+efficacy+of+the+heat+killing+of+Mycobacterium+tuberculosis%5BTitle%5D)+AND+%22J+Clin+Pathol%22%5BJournal%5D)">The efficacy of the heat killing of Mycobacterium tuberculosis.</a> J Clin Pathol. 55 (10), 778-779 (2002).</li>
<li>Dippenaar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exploring+the+potential+of+Oxford+Nanopore+Technologies+sequencing+for+Mycobacterium+tuberculosis+sequencing%3A+An+assessment+of+R10+flowcells+and+V14+chemistry%5BTitle%5D)+AND+%22PLOS+ONE%22%5BJournal%5D)">Exploring the potential of Oxford Nanopore Technologies sequencing for Mycobacterium tuberculosis sequencing: An assessment of R10 flowcells and V14 chemistry.</a> PLOS ONE. 19 (6), e0303938(2024).</li>
<li>Chang, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metagenomic+DNA+sequencing+to+quantify+Mycobacterium+tuberculosis+DNA+and+diagnose+tuberculosis%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Metagenomic DNA sequencing to quantify Mycobacterium tuberculosis DNA and diagnose tuberculosis.</a> Sci Rep. 12, 16972(2022).</li>
<li>Forbes, J. D., Knox, N. C., Ronholm, J., Pagotto, F., Reimer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metagenomics%3A+The+next+culture-independent+game+changer%5BTitle%5D)+AND+%22Front+Microbiol%22%5BJournal%5D)">Metagenomics: The next culture-independent game changer.</a> Front Microbiol. 8, 1069(2017).</li>
<li>Marshall, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+of+isolation+and+identification+of+non-tuberculous+mycobacteria+from+environmental+samples%3A+A+scoping+review%5BTitle%5D)+AND+%22Tuberculosis%22%5BJournal%5D)">Methods of isolation and identification of non-tuberculous mycobacteria from environmental samples: A scoping review.</a> Tuberculosis. 138, 102291(2023).</li>
</ol>

The authors have no conflicts of interest to disclose.

Representative results demonstrate the utility of the CTAB method in extracting DNA from mycobacterial species and addressing the challenges associated with extracting DNA from these organisms. Mycobacteria&#160;are well known for their complex and lipid-rich cell walls2. The current results highlight several critical aspects that contribute to the success of this technique as it delivers high-concentration, high-quality intact DNA that should be suitable for downstream applications such as PCR, s...

Extracting high-quality DNA is a fundamental step in molecular biology, essential for downstream applications such as PCR, whole genome sequencing, and other genomic studies. For Mycobacterium, a genus that includes both pathogenic species, such as Mycobacterium tuberculosis (M.tuberculosis), and non-pathogenic environmental species like Mycobacterium smegmatis (M. smegmatis), DNA extraction can be challenging due to their thick, lipid-rich, and hydrophobic cell walls1,2,3. This tough cell wall necessitates utilizing specialized lysis methods to disrupt the cells and effectively release genomic DNA.
The cetyltrimethylammonium bromide (CTAB) method has proven reliable for isolating high-quality DNA from various mycobacterial species4,5. CTAB, a cationic surfactant, binds to polysaccharides and other cell components, facilitating their removal during the extraction process6. Even with difficult samples, the CTAB-based protocol ensures the efficient isolation of intact and pure DNA, particularly when combined with enzymatic lysis, heat treatment, and organic solvent extractions7,8. The CTAB method has played a crucial role in efficient DNA extraction from mycobacterial species, specifically for research involving whole-genome sequencing, metagenomics, and molecular diagnostics, which require pure and high-quality DNA input material9,10. Compared to other DNA extraction techniques such as bead-beating or commercial kits, the CTAB protocol presented here offers a distinct advantage in preserving DNA integrity, an essential factor for long-read sequencing and epigenetic applications. While earlier studies have reported lower DNA yields with CTAB relative to mechanical disruption methods, they also emphasize its ability to minimize DNA shearing and maintain DNA integrity11,12. To address limitations in purity and yield, the current protocol has been optimized with reduced incubation times and improved consistency across diverse sample types. This refinement was supported by findings from Opperman et al., who demonstrated that CTAB-extracted DNA from Mycobacterium avium cultures was of sufficient concentration and quality for downstream applications such as 16S rRNA sequencing and line probe assays, further supporting the application of the CTAB protocol as a versatile and scalable tool for mycobacterial research, particularly in studies requiring intact genomic DNA for advanced molecular analyses13. Additionally, next-generation sequencing (NGS) generally requires 100-1000 ng of high-quality genomic DNA14 and in the case of long-read sequencing, such as PacBio sequencing, requires &#8805;5 &#181;g of High-quality DNA15, thus when large amounts of high-quality and intact DNA are required, CTAB remains an effective method16.
Establishing efficient, consistent, and effective DNA extraction methodologies from tough-to-lyse microbes is necessary to ensure reproducibility and reliability across laboratories, particularly for strain identification and epidemiological studies5,7. This protocol provides details for extracting whole-genome DNA from Mycobacteria using the CTAB method. The current protocol aims to enable researchers to achieve consistent and reliable results in their molecular biology workflows by addressing the pitfalls and overcoming the challenges posed by the distinctive mycobacterial cell wall17.

<table><tbody><tr><td>1 mL pipette tips</td><td>Any supplier</td><td></td><td></td></tr><tr><td>10 &amp;micro;L pipette tips</td><td>Any supplier</td><td></td><td></td></tr><tr><td>10% Sodium Dodecyl Sulfate (SDS)</td><td>Merck (Sigma-Aldrich)</td><td>71736-100 ML</td><td></td></tr><tr><td>15 mL polypropylene tubes</td><td>Any supplier</td><td></td><td></td></tr><tr><td>2 ml tubes and 1.5 ml tubes</td><td>Any supplier</td><td></td><td></td></tr><tr><td>200 &amp;micro;l pipette tips</td><td>Any supplier</td><td></td><td></td></tr><tr><td>2-PROPANOL, BIOREAGENT, FOR MOLECULAR B&amp;amp;</td><td>Merck (Sigma-Aldrich)</td><td>I9516-1L&amp;nbsp;</td><td>Store in -20 &amp;deg;C</td></tr><tr><td>37&amp;deg;C incubator</td><td>Any supplier</td><td></td><td></td></tr><tr><td>5 M NaCl</td><td>Merck (Sigma-Aldrich)</td><td>S6546 - 100 ML</td><td></td></tr><tr><td>65&amp;deg;C incubator</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Agarose gel</td><td>Merck (Sigma-Aldrich)</td><td></td><td></td></tr><tr><td>Bio-Rad UV gel documentation system,</td><td>BioRad</td><td></td><td></td></tr><tr><td>Centrifuge (for 15 ml tubes oe 50 ml) and Microfuge (for 2 ml tubes)</td><td>Any supplier</td><td></td><td></td></tr><tr><td>CHLOROFORM FOR ANALYSIS EMPARTA&amp;reg; ACS</td><td>Merck (Sigma-Aldrich)</td><td>1070242500</td><td></td></tr><tr><td>Chloroform/isoamyl alcohol (24:1)</td><td></td><td></td><td>In a fume hood, mix 384 ml Chloroform and 16 ml Isoamyl alcohol</td></tr><tr><td>CTAB/NaCl solution</td><td>Merck (Sigma-Aldrich)</td><td>52370-100 G</td><td>Dissolve 4.1 g NaCl in 80 ml distilled water. While stirring, add 10 g CTAB. Heat solution in 65&amp;deg;C incubator. Adjust volume to 100 ml with distilled water. Store at room temperature for up to 6 months.</td></tr><tr><td>DNA 1 Kb ladder</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>ETHYL ALCOHOL, PURE, 200 PROOF, FOR MOL&amp;amp;</td><td>Merck (Sigma-Aldrich)</td><td>E7023-500ML</td><td>Mix 75 ml Absolute ethanol and 25 ml distilled water</td></tr><tr><td>Lysozyme (10 mg/ml)</td><td>Merck (Sigma-Aldrich)</td><td>10837059001</td><td>Reconstitute lyophilized lysozyme (brought to room temperature) with distilled water to 10 mg/ml</td></tr><tr><td>Middlebrook 7H9 media</td><td>Merck (Sigma-Aldrich)</td><td>M0178</td><td>Follow the manufacturer&#039;s guidelines for preparing the liquid media</td></tr><tr><td>MIDDLEBROOK OADC ENRICHMENT 6 x 100 ml F&amp;nbsp;</td><td>BD</td><td>212240</td><td></td></tr><tr><td>Mycobacteria Growth Indicator Tube (MGIT) media</td><td>BD</td><td>245122</td><td></td></tr><tr><td>NanoDrop system</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>P10 pipette</td><td>Any supplier</td><td></td><td></td></tr><tr><td>P1000 pipette</td><td>Any supplier</td><td></td><td></td></tr><tr><td>P200 pipette</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Proteinase K, recomb, PCR grd 2x250mg&amp;nbsp;</td><td>Merck (Sigma-Aldrich)</td><td>3115801001</td><td>Reconstitute vial of lyophilised Proteinase K with distilled water to 10 mg/ml. Freeze aliquots in 2 ml tubes at -20 &amp;deg;C</td></tr><tr><td>SYBR-safe</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Tris/EDTA (TE) buffer pH 8.0</td><td>Merck (Sigma-Aldrich)</td><td>8890-OP or 382499997</td><td></td></tr><tr><td>Vortex</td><td>Any supplier</td><td></td><td></td></tr></tbody></table>

whole-genome deoxyribonucleic acid extraction from mycobacterium species via the cetyltrimethylammonium bromide technique

Whole-Genome Deoxyribonucleic Acid Extraction from Mycobacterium Species via the Cetyltrimethylammonium Bromide Technique

Efficient and reliable DNA extraction is a fundamental step in molecular biology, providing high-quality genetic material for downstream applications such as PCR, sequencing, and genomic analyses. However, the unique cell wall composition of mycobacteria, rich in lipids and mycolic acids, poses significant challenges for efficient DNA isolation. The cetyltrimethylammonium bromide (CTAB) method is widely employed due to its efficiency in extracting high-purity DNA from mycobacterial species, including both fast- and slow-growing strains. The technique involves enzymatic disruption of bacterial cells using lysozyme and proteinase K, followed by the lysis of cell components using CTAB, which selectively binds and forms a complex with polysaccharides and proteins. DNA purification and concentration are achieved by organic solvent extraction and ethanol precipitation. According to our data, the CTAB protocol yields high quantities of DNA (200 - 1000 ng/&#181;L) with high quality and minimal contamination as proven by gel electrophoresis and NanoDrop analysis. The DNA extracted from different mycobacterial species is of high purity and is suitable for various molecular studies. This study demonstrates the CTAB method's procedural steps, highlighting its utility in overcoming the unique challenges associated with mycobacterial DNA extraction.

This DNA extraction protocol using the CTAB method (Figure 1) was assessed across various mycobacterial species such as M. tuberculosis, M. abscessus, and M. chelonea. DNA was extracted and compared regarding yield, purity, and integrity of the extracted DNA across samples.
DNA concentration varied across the mycobacterial species and ranged between 190-600 ng/µL and &gt;1000 ng/µL in some instances, indicating that CTAB extracts high-yi...

This protocol describes the CTAB method for extracting high-quality DNA from mycobacteria, overcoming challenges posed by their tough, mycolic acid-rich cell walls. The process involves enzymatic digestion, cell lysis with CTAB, and DNA purification through organic extraction and ethanol precipitation. This method produces DNA suitable for molecular studies and is reliable for mycobacterial research.

Efficient and reliable DNA extraction is a fundamental step in molecular biology, providing high-quality genetic material for downstream applications such ...

Our research centers on the genetic characterization of mycobacterial species and the complex nature of mycobacterial diseases in human. We place particular emphasis on nontuberculosis mycobacteria and their interactions with macrophage during coinfection with other species, especially microbacteria tuberculosis. Additionally, we focus on the speciation of pathogenic mycobacteria, identification on virulence-associated genes, and the single nucleotide polymorphism linked to drug resistance.Due to the thick cell wall, lipidic and hydrophobic cell walls of mycobacteria DNA extraction has been challenging and requires specialized lysis techniques to break them down and efficiently release the genomic DNA. In our protocol, we are addressing DNA extraction from mycobacteria species using the CTAB method as a robust and effective method to produce high-quality DNA that is suitable for all genome sequencing and other molecular techniques. To begin, heat kill the liquid culture taken in a 15-milliliter tube at 80 degrees Celsius for one hour.Then place the 15 milliliter tube in the centrifuge and spin at 3, 220 G for 15-30 minutes at room temperature. Using a sterile pipette, discard the clear supernatant, ensuring that all the media is removed. Resuspend the culture pellet thoroughly in 300 microliters of Tris-EDTA buffer, and transfer the resuspended pellet into a 2-milliliter tube.Now, add 100 microliters of lysozyme solution at a concentration of 10 milligrams per milliliter to the tube and mix by pipetting up and down five times. Tap the tube gently to ensure uniform dispersion and place the tube in a rotary incubator at 37 degrees Celsius and incubate overnight. The following day, prepare a mixture of 5 microliters of proteinase K at a concentration of 10 milligrams per milliliter and 70 microliters of 10%SDS.Add 75 microliters of this mixture to each sample. Mix the sample by tapping the tube and incubate at 65 degrees Celsius for 10 minutes, intermittently inverting or tapping the tube to mix. Next, add 100 microliters of 5 molar sodium chloride to the sample, followed by 100 microliters of preheated CTAB sodium chloride solution preheated to a temperature of 65 degrees Celsius to the sample.Mix the sample by tapping until the solution becomes milky. Place the tube in an incubator at 65 degrees Celsius for 10 minutes, intermittently inverting or tapping the tube to mix. Now, add an equal volume, approximately 675 microliters of chloroform isoamyl alcohol solution in a ratio of 24:1 to the sample and mix by tapping.Place the sample in a centrifuge and spin at 12, 000 G for 10 minutes at room temperature. Then, using a pipette, carefully aspirate between 550-600 microliters of the aqueous top phase into sterile 1.5-milliliter tubes and label them appropriately. Add 550-600 microliters of ice cold isopropanol to each tube and mix the contents by inverting the tube several times.Place the tube in a freezer set to 20 degrees Celsius and incubate for 30 minutes to one hour. Next, centrifuge the tube at the highest speed, approximately 21, 130 G for 30 minutes at room temperature to pellet the insoluble DNA. Using a pipette, aspirate the supernatant from the front side of the tube without disturbing the DNA pellet.Now, add 1, 000 microliters of ice cold 75%ethanol to the pellet, and mix the sample by inverting the tube several times. Centrifuge the sample at 12, 000 G for 30 minutes in the same orientation as previously used. After centrifugation, aspirate or decant all ethanol without disturbing the pellet.Allow the tube to air dry at room temperature overnight or for at least 30 minutes. Then, add between 25-50 microliters of Tris-EDTA buffer at pH 8 to resuspend the DNA, and place the tube at 4 degrees Celsius overnight. After quality control assessment, place the tube in a freezer set to 80 degrees Celsius for long-term storage.DNA yield obtained using the CTAB extraction method ranged between 190 nanograms per microliter and 600 nanograms per microliter. The 260/280 absorbance ratio ranged between 1.9 and 2.0, and the 260/230 ratio ranged between 1.8 and 2.2, indicating high purity DNA. A single absorbance peak was detected at 260 nanometers.Agarose gel electrophoresis of high quality samples showed a distinct intact high molecular weight DNA band with minimal degradation. In some nontuberculosis mycobacterium species, DNA yield was less than 50 nanograms per microliter and purity ratios fell below the ideal range, indicating contamination.

whole-genome-deoxyribonucleic-acid-extraction-from-mycobacterium

Research

JoVE Journal

Biology

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 μm Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material.  This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

 DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification.  This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Extraction of High Molecular Weight DNA from Microbial Mats

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures.  Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism.  Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

We present the process of isolating, propagating, and characterizing hydrocarbon-degrading bacteria from aquatic habitats. The protocol outlines bacterial isolation, identification by the 16S rRNA method, and testing of their hydrocarbon-degrading potential. This article would help researchers in characterizing microbial biodiversity in environmental samples, and specifically screen for microbes with bioremediation potential.

Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats

This protocol shows bead-beating combined with DNA capture bead purification provides a fast and consistent method for extracting DNA from Mycobacterium tuberculosis samples, making it an effective choice for next-generation sequencing applications.

Mycobacterial DNA Extraction using Bead Beating in Custom Buffer Followed by NGS Workflow

We present an optimized method for DNA extraction from decontaminated sputum using a matrix-based extraction method combined with a magnetic bead purification for downstream targeted next-generation sequencing of Mycobacterium tuberculosis.

Matrix-based DNA Extraction for Targeted Next-Generation Sequencing on Decontaminated Sputum Samples

Here, we present a DNA microarray chip method for identifying Mycobacterium species, which will improve diagnostic accuracy and efficiency of related diseases for clinical use.

Identification of Mycobacterium Species by DNA Microarray Chip Method

Learn the CTAB method for extracting high-quality DNA from Mycobacterium species with robust cell walls. This protocol yields pure genomic DNA suitable for sequencing and molecular studies.

Whole-Genome Deoxyribonucleic Acid Extraction from Mycobacterium Species via the Cetyltrimethylammonium Bromide Technique

Explore More Videos