eDNA extraction: phenol-chloroform-isoamyl alcohol DNA purification from filters stored in Longmire buffer v1

10.17504/protocols.io.bx4apqse ◽

2021 ◽

Author(s):

Ana Laca ◽

Abigail Wells ◽

Linda Park

Keyword(s):

Dna Extraction ◽

Extraction Method ◽

Isoamyl Alcohol ◽

Dna Purification ◽

Phase Lock ◽

Dna Extraction Method ◽

Aqueous Layer

This is an organic DNA extraction method for filters preserved in 2 ml of Longmire buffer that uses a phase lock to allow easy decanting of the aqueous layer instead of pipetting.

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A modified SDS-based DNA extraction method from raw soybean

Bioscience Reports ◽

10.1042/bsr20182271 ◽

2019 ◽

Vol 39 (2) ◽

Cited By ~ 10

Author(s):

Yimiao Xia ◽

Fusheng Chen ◽

Yan Du ◽

Chen Liu ◽

Guanhao Bu ◽

...

Keyword(s):

Dna Extraction ◽

Extraction Method ◽

Pcr Amplification ◽

Isoamyl Alcohol ◽

Monitoring Methods ◽

Detection Techniques ◽

Dna Yield ◽

Seed Flour ◽

Dna Extraction Method ◽

Lysis Buffer

Abstract Soybean is the most important genetically modified (GM) oilseed worldwide. Regulations relating to the approval of biotech soybean varieties and product labeling demand accurate and reliable detection techniques to screen for GM soya. High-quality extracted DNA is essential for DNA-based monitoring methods. Thus, four widely used protocols (SDS, CTAB, DP305, and DNeasy Plant Mini Kit) were compared in the present study to explore the most efficient DNA extraction method for raw soya matrix. The SDS-based method showed the highest applicability. Then crucial factors influencing DNA yield and purity, such as SDS lysis buffer component concentrations and organic compounds used to isolate DNA, were further investigated to improve the DNA obtained from raw soybean seeds, which accounts for the innovation of this work. As a result, lysis buffer (2% SDS (w/v), 150 mM NaCl, 50 mM Tris/HCl, 50 mM EDTA, pH 8.0) and organic reagents including chloroform/isoamyl alcohol (24:1, v/v) (C: I), isopropanol, and ethanol corresponding to the extraction and first and second precipitation procedures, respectively, were used in the optimized SDS method. The optimized method was verified by extracting approximately 2020–2444 ng DNA/mg soybean with A260/280 ratios of 1.862–1.954 from five biotech and non-biotech soybean varieties. Only 0.5 mg of soya was required to obtain enough DNA for PCR amplification using the optimized SDS-based method. These results indicate that the screening protocol in the present study achieves the highest suitability and efficiency for DNA isolation from raw soya seed flour.

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A comparison of different DNA extraction methods and molecular techniques for the detection and identification of foodborne pathogens

AIMS Microbiology ◽

10.3934/microbiol.2021019 ◽

2021 ◽

Vol 7 (3) ◽

pp. 304-319

Author(s):

Spyridon Andreas Papatheodorou ◽

◽

Panagiotis Halvatsiotis ◽

Dimitra Houhoula ◽

Keyword(s):

Dna Extraction ◽

Foodborne Pathogens ◽

Extraction Method ◽

Pathogenic Bacteria ◽

Low Cost ◽

Isoamyl Alcohol ◽

Extraction Methods ◽

Molecular Techniques ◽

Bacterial Dna ◽

Dna Extraction Method

<abstract> <p>Foodborne infections continue to plague Europe. Food safety monitoring is in crisis as the existing techniques for detecting pathogens do not keep up with the global rising of food production and consumption. Thus, the development of innovative techniques for detecting and identifying pathogenic bacteria has become critical. The aim of the present study was firstly to develop an innovative simple and low cost method of extracting bacterial DNA from contaminated food and water samples with <italic>Salmonella enteric</italic> subsp. <italic>enteric</italic> serovar Typhimurium and <italic>Listeria monocytogenes</italic> and its comparison with two commercial DNA extraction kits (Qiagen, Macherey-Nagel). Finally, pathogens' detection using two molecular techniques (PCR-electrophoresis, LAMP), in order to evaluate the best combination of DNA extraction and identification based on their sensitivity, cost, rapidity and simplicity. Considering the above criteria, among them, best was proved an in-house bacterial DNA extraction method, based on the chloroform-isoamyl alcohol protocol, with certain modifications. This technique showed statistically similar results in terms of sensitivity, compared to the commercial kits, while at the same time maintained high rapidity and much lower cost. Lastly, between the molecular techniques, LAMP was found more promising considering its simplicity, high rapidity and sensitivity. Conclusively, the in-house DNA extraction method along with the LAMP technique, was proven to be the best among the presented combinations.</p> </abstract>

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Fecal genomic DNA extraction method impacts outcome of MinION based metagenome profile of tuberculosis patients.

10.1101/2021.11.15.21266154 ◽

2021 ◽

Author(s):

Sukanya Sahu ◽

Sandeep Kaushik ◽

Bidhan Goswami ◽

Arunabha Dasgupta ◽

Hritusree Guha ◽

...

Keyword(s):

Microbial Diversity ◽

Dna Extraction ◽

Gut Microbiome ◽

Extraction Method ◽

Critical Role ◽

Isoamyl Alcohol ◽

Fecal Samples ◽

Sequencing Platform ◽

Dna Extraction Method ◽

Generation Sequencing

In the present era, emergence of next generation sequencing approaches has revolutionized the field of gut microbiome study. However, the adopted DNA extraction step used in metagenomics experiments and its efficiency may play a critical role in their reproducibility and outcome. In this study, fecal samples from active and non-tuberculosis subjects (ATB/NTB, n=7) were used. Fecal samples of a subgroup of these subjects were subjected to Mechanical enzymatic lysis (MEL) and Phenol: Chloroform: Isoamyl Alcohol (PCIA) methods of DNA extraction and a third-generation sequencing platform i.e., MinION was employed for microbiome profiling. Findings of this study demonstrated that DNA extraction method significantly impacts the DNA yield and microbial diversity. Irrespective of the adopted method of DNA extraction, ATB patients showed altered microbial diversity compared to NTB controls. Also, the fecal microbial diversity details are better captured in samples processed by MEL method and may be suitable to be adopted for high-throughput gut microbiome studies.

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EFFICIENCY OF TRADITIONAL DNA EXTRACTION METHOD IN PCR DETECTION OF PORCINE DNA IN MEAT MIXTURES

Jurnal Teknologi ◽

10.11113/jt.v82.14636 ◽

2020 ◽

Vol 82 (5) ◽

Author(s):

Zaliha Suadi ◽

Lesley Maurice Bilung ◽

Kasing Apun ◽

Aida Azrina Azmi

Keyword(s):

Dna Extraction ◽

Extraction Method ◽

Isoamyl Alcohol ◽

Cost Effective ◽

Raw Meat ◽

Dna Extraction Method ◽

Meat Samples ◽

Species Specific ◽

Cyt B Gene

Through the advancement of biotechnology, DNA-based methods are the most effective techniques in species identification, as they are rapid and have higher stability in harsh conditions compared to protein-based methods. This study was conducted to determine the efficiency of the traditional DNA extraction method, phenol/chloroform/isoamyl alcohol (PCIA), and comparing it with the commercially available kit by evaluating the purity, concentration, and suitability for amplification of porcine DNA in raw chicken and beef mixtures. The quantity and quality of the DNA extracts were assessed using a UV-Vis spectrophotometer. Polymerase chain reaction (PCR) was performed using species-specific primers targeting mitochondrial DNA cytochrome b (cyt b) gene of chicken (227-bp), beef (274-bp), and pork (398-bp), to confirm the template usability and quality of the DNA extracts. High DNA concentrations and purity were obtained from meat samples extracted using the PCIA method. The visualization of pork DNA on 2% agarose gel was able to detect pork contamination in raw meat mixtures up to minute proportion (1%). The existence of pork in chicken and beef was indicated with the presence of a specific 398-bp DNA band. Thus, the PCIA method can be recommended as a cost-effective and an excellent alternative to more expensive extraction kits in detecting pork DNA in raw meat mixtures.

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Comparison of DNA Concentration and Purity of Animal Blood Extracted using Different DNA Extraction Kits

Malaysian Journal of Science Health & Technology ◽

10.33102/mjosht.v2i.42 ◽

2018 ◽

Vol 2 ◽

Author(s):

Mohamed Sabri Esa ◽

Nur Huda Faujan ◽

Haitham Abdullah Rajab ◽

Maryam Mohamed Rehan ◽

Norlelawati Arifin ◽

...

Keyword(s):

Dna Extraction ◽

Genomic Dna ◽

Extraction Method ◽

Bos Taurus ◽

Dna Purification ◽

Chain Reaction ◽

Animal Blood ◽

Dna Concentration ◽

Dna Extraction Method ◽

Polymerase Chain

The efficiency of DNA extraction from whole blood using appropriate method is very important for molecular analysis. Therefore, the aim of this study was to compare the purity and concentration of DNA extraction method from bovine (Bos taurus), chicken (Gallus gallus), and porcine (Sus scrofa) blood. The DNA of blood samples was extracted using three types of kit, namely InnuPREP Blood DNA Mini Kit, Wizard Genomic DNA Purification Kit, and QIAamp DNA Blood Mini Kit. The results showed that blood DNA extracted using QIAamp DNA Blood Mini Kit was found to be the most effective and consistently produced high concentrated and pure DNA for three animal samples. The purity of DNA ranged from 1.73 ± 0.05 Å to 1.94 ± 0.21 Å and the range of blood DNA concentration extracted using the QIAamp DNA were between 13.73 ± 2.11 and 25.01 ± 2.08 ng/?l. However, the blood DNA of porcine was not successfully extracted using InnuPREP Blood DNA Mini Kit and Wizard® Genomic DNA Purification Kit. These results were very crucial for the subsequent use of amplification using polymerase chain reaction (PCR) and to facilitate accurate detection in further analysis.

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Sensitivity detection of Abacavir in human through SNP detection of HLA-B*5701 allele

International Journal of Bioassays ◽

10.21746/ijbio.2016.08.006 ◽

2016 ◽

Vol 5 (08) ◽

pp. 4754

Author(s):

Tanushree Mitra* ◽

Shivshankar Kumdale ◽

Sameer Chowdhary ◽

Amol D. Raut

Keyword(s):

Blood Sample ◽

Dna Extraction ◽

Genomic Dna ◽

Extraction Method ◽

Snp Detection ◽

Dna Extraction Method ◽

Sensitivity Detection

The main objective of this study was to make sure whether randomly taken 12 samples were sensitive to abacavir. The genomic DNA from 12 blood sample were extracted by phenol chloroform DNA extraction method, extracted genomic DNA were amplified and sequenced, thereafter SNPs were detected. Every sample had shown the presence of normal base at SNP position. This study indicated, those randomly taken 12 patients were sensitive to abacavir, so they can consume abacavir if they get infected with HIV.

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