DNA extraction

2018 ◽  
Author(s):  
Pierre Taberlet ◽  
Aurélie Bonin ◽  
Lucie Zinger ◽  
Eric Coissac
Keyword(s):  
Dna Extraction ◽  
Phosphate Buffer ◽  
Environmental Samples ◽  
Soil Samples ◽  
Mobile Laboratory ◽  
Extraction Protocol ◽  
Soil Sediment ◽  
Total Dna ◽  
Practical Constraints ◽  
Detailed Protocol

Chapter 5 “DNA extraction” focuses on the particularities and practical constraints associated with the isolation of eDNA from environmental samples. The extraction protocol is indeed crucial in eDNA studies, as it will determine whether extracellular, intracellular, or total DNA is targeted. Chapter 5 describes the main advantages and limitations of the most popular extraction kits aimed at obtaining DNA from soil, sediment, litter, feces, or water. It provides a detailed protocol for DNA extraction from soil samples using a saturated phosphate buffer. This protocol has been optimized for an easy implementation in the field using a mobile laboratory, so the material and consumables necessary are also listed.

scholarly journals Improved Protocol for DNA Extraction from Subsoils Using Phosphate Lysis Buffer

2020 ◽  
Vol 8 (4) ◽  
pp. 532 ◽  
Author(s):  
Victor Guerra ◽  
Lukas Beule ◽  
Ena Lehtsaar ◽  
Hui-Ling Liao ◽  
Petr Karlovsky
Keyword(s):  
Dna Extraction ◽  
Phosphate Buffer ◽  
Sodium Dodecyl ◽  
Clay Content ◽  
Cost Effective ◽  
Rrna Genes ◽  
Soil Biology ◽  
Agricultural Field ◽  
Extraction Protocol ◽  
18S Rrna Genes

As our understanding of soil biology deepens, there is a growing demand for investigations addressing microbial processes in the earth beneath the topsoil layer, called subsoil. High clay content in subsoils often hinders the recovery of sufficient quantities of DNA as clay particles bind nucleic acids. Here, an efficient and reproducible DNA extraction method for 200 mg dried soil based on sodium dodecyl sulfate (SDS) lysis in the presence of phosphate buffer has been developed. The extraction protocol was optimized by quantifying bacterial 16S and fungal 18S rRNA genes amplified from extracts obtained by different combinations of lysis methods and phosphate buffer washes. The combination of one minute of bead beating, followed by ten min incubation at 65°C in the presence of 1 M phosphate buffer with 0.5% SDS, was found to produce the best results. The optimized protocol was compared with a commonly used cetyltrimethylammonium bromide (CTAB) method, using Phaeozem soil collected from 60 cm depth at a conventional agricultural field and validated on five subsoils. The reproducibility and robustness of the protocol was corroborated by an interlaboratory comparison. The DNA extraction protocol offers a reproducible and cost-effective tool for DNA-based studies of subsoil biology.

Sample collection and eDNA extraction from Sterivex filter units v1

2021 ◽  
Author(s):  
Oscar E Chiang ◽  
Pedro Inostroza
Keyword(s):  
Dna Extraction ◽  
Environmental Samples ◽  
Dna Analysis ◽  
Water Systems ◽  
Sample Collection ◽  
Extraction Protocol ◽  
Water Collection ◽  
Sterile Filter

The following workflow covers several steps in the DNA analysis of environmental samples, from the water collection to the analysis back in the lab. The samples can be taken from several water systems (i.e. sea, lakes, rivers, streams) and collected in triplicate (1 L) in Sterivex sterile filter units (Merck, cat. no. SVGP01050). The DNA extraction protocol modifies the Dneasy PowerWater Sterivex kit (Qiagen, cat. no. 14600-50-nf).

Total DNA extraction from microalgae strain samples using NucleoSpin Plant modified kit (Macherey Nagel) v1

2021 ◽  
Author(s):  
Sarah Romac
Keyword(s):  
Dna Extraction ◽  
Extraction Protocol ◽  
Microbiome Diversity ◽  
Total Dna

This DNA extraction protocol allows to get both eukaryotic and prokaryotic DNA from microalgae strains, so the microbiome diversity can be studied in cultures by using this protocol.

scholarly journals Development of Rapid and Less Hazardous Plant DNA Extraction Protocol using Potassium Phosphate Buffer

2021 ◽  
Vol 24 (12) ◽  
pp. 1309-1315
Author(s):  
Jamsari Jamsari ◽  
Muhammad Arif Setia ◽  
Bastian Nova ◽  
Lily Syukriani ◽  
Siti Nur Aisyah ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Phosphate Buffer ◽  
Potassium Phosphate ◽  
Potassium Phosphate Buffer ◽  
Plant Dna ◽  
Extraction Protocol

scholarly journals Optimized DNA-based identification of Toxocara spp. eggs in soil and sand samples

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Wojciech Jarosz ◽  
Jean-Francois Durant ◽  
Leonid Mwana Wa Bene Irenge ◽  
Renata Fogt-Wyrwas ◽  
Hanna Mizgajska-Wiktor ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Environmental Samples ◽  
Limit Of Detection ◽  
Toxocara Canis ◽  
Extraction Methods ◽  
Soil Samples ◽  
Toxocara Cati ◽  
Flow Process ◽  
Spiked Soil ◽  
Set Up

Abstract Background Toxocara canis and Toxocara cati are globally distributed roundworms and causative agents of human toxocariasis, via ingestion of Toxocara eggs. Control of Toxocara infections is constrained by a lack of sensitive methods for screening of animal faeces and environmental samples potentially contaminated by Toxocara eggs. In this work, a pre-analytical method for efficient extraction of DNA from Toxocara eggs in environmental samples was set up using our previously validated T. canis- and T. cati-specific quantitative real-time polymerase chain reaction (qPCR). For this purpose, the influence of different methods for egg lysis, DNA extraction and purification for removal of PCR inhibitors were assessed on environmental samples. Methods To select the best egg disruption method, six protocols were compared on pure T. canis egg suspensions, including enzymatic lysis and thermal or mechanical disruption. Based on the selected best method, an analytical workflow was set up to compare two DNA extraction methods (FastDNA™ SPIN Kit for Soil versus DNeasy® PowerMax® Soil Kit) with an optional dilution and/or clean-up (Agencourt® AMPure®) step. This workflow was evaluated on 10-g soil and 10-g sand samples spiked with egg suspensions of T. canis (tenfold dilutions of 104 eggs in triplicate). The capacity of the different methods, used alone or in combination, to increase the ratio of positive tests was assessed. The resulting optimal workflow for processing spiked soil samples was then tested on environmental soil samples and compared with the conventional flotation-centrifugation and microscopic examination of Toxocara eggs. Results The most effective DNA extraction method for Toxocara eggs in soil samples consisted in the combination of mechanical lysis of eggs using beads, followed by DNA extraction with the DNeasy® PowerMax® Soil Kit, and completed with an additional DNA clean-up step with AMPure® beads and a sample DNA dilution (1:10). This workflow exhibited a limit of detection of 4 and 46 T.canis eggs in 10-g sand and 10-g soil samples, respectively. Conclusions The pre-analytical flow process developed here combined with qPCR represents an improved, potentially automatable, and cost-effective method for the surveillance of Toxocara contamination in the environment. Graphical Abstract

OPTIMATION OF DNA EXTRACTION PROTOCOL OF Elaeidobius kamerunicus

2018 ◽  
Vol 24 (2) ◽  
pp. 77-86
Author(s):  
Sri Wening ◽  
Agus Eko Prasetyo ◽  
Tjut Ahmad Perdana Rozziansha ◽  
Agus Susanto
Keyword(s):  
Dna Fingerprinting ◽  
Dna Extraction ◽  
Oil Palm ◽  
Fruit Set ◽  
Fragment Length Polymorphism ◽  
Basic Knowledge ◽  
Biological Study ◽  
Palm Fruit ◽  
Extraction Protocol ◽  
Elaeidobius Kamerunicus

African pollination weevil (Elaeidobius kamerunicus Faust) has an important role in the productivity of Indonesian oil palm plantation. Up to now, there has not been a comprehensive biological study of the species at molecular level. The basic knowledge is very useful for exploitation of the weevil for effective oil palm fruit set development. This research aimed to obtain DNA extraction protocol of E. kamerunicus for DNA fingerprinting of the species. Results showed that using a DNA extraction kit,material disruption by using micro pestle resulted the highest quantity of DNA, while there were no significant differences of resulted DNA quantity among treatments using tissue lyser for material disruption. DNA extracted by using micro pestle or tissue lyser for material disruption is adequate for DNA fingerprinting using AFLP (Amplified Fragment Length Polymorphism) and sequencing techniques.

A modified DNA extraction protocol and its utility in seed genetic purity assessment

2011 ◽  
Vol 39 (1) ◽  
pp. 236-242 ◽  
Author(s):  
S.N. Sharma ◽  
V. Kumar ◽  
G. Singh ◽  
R. Sharma
Keyword(s):  
Dna Extraction ◽  
Genetic Purity ◽  
Purity Assessment ◽  
Extraction Protocol ◽  
Modified Dna

Quantification of Tire-Tread Particles Using Extractable Organic Zinc as Tracer

1999 ◽  
Vol 72 (5) ◽  
pp. 969-977 ◽  
Author(s):  
Patrik Fauser ◽  
Jens Christian Tjell ◽  
Hans Mosbaek ◽  
Kim Pilegaard
Keyword(s):  
Atomic Absorption Spectrometry ◽  
Atomic Absorption ◽  
Environmental Samples ◽  
High Sensitivity ◽  
Absorption Spectrometry ◽  
Soil Samples ◽  
New Method ◽  
Organic Zinc

Abstract A method for identifying and quantifying tire-tread particles in the environment has been developed. It is based on the measurement of extractable organic zinc. The high sensitivity of atomic absorption spectrometry (AAS) with a heated graphite atomizer (HGA) permits assessment of submilligram amounts of tire debris in environmental samples. The analysis is performed on aerosol and soil samples. This new method is more accurate and faster than the previously reported IR method.

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