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Optimisation of protocol for effective detachment and selective recovery of the representative bacteria for extraction of metagenomic DNA from Eucalyptus spp. woodchips
- Source :
- Journal of Microbiological Methods. 148:155-160
- Publication Year :
- 2018
- Publisher :
- Elsevier BV, 2018.
-
Abstract
- For some environments such as planktonic/aqueous environments, the separation of bacteria cells from eukaryotic cells prior to DNA extraction using filtration is relatively straightforward. However, for woodchips, the bacteria are attached/embedded within the wood matrix, which prevents easy removal of bacterial cells. In this study, a method for the selective extraction of DNA from bacteria inhabiting Eucalyptus spp. woodchips has been developed. The objective was to compare milled and unmilled woodchips processed via three detachment methods, viz., sonication, vortexing and shaking followed by filtration using Teflon filters according to three relevant criteria: DNA yield, DNA purity and quality of DNA. Highest DNA yield was obtained by milling and vortexing for 10 min (77.50 ± 5.17 ng/μl), followed by milling and vortexing for 2 min (61.00 ± 6.56 ng/μl), unmilled and vortexing for 10 min (38.67 ± 5.17 ng/μl) and milled and shaking for 2 h (31.62 ± 5.17 ng/μl). The lowest DNA yield was obtained by using unmilled woodchips and 5 min of sonication treatment (7.00 ± 1.22 ng/μl). There was no significant difference in DNA purity for milled or unmilled woodchips processed via the three detachment methods. Duration of cell detachment treatment did not significantly influence DNA yield and purity. Following optimisation experiments, it was possible to extract bacterial DNA using milled woodchips and 10 minute vortexing devoid of DNA from the host background and other associated eukaryotes and of sufficient quality and quantity for metagenomic analysis.
- Subjects :
- DNA, Bacterial
0301 basic medicine
Microbiology (medical)
Sonication
030106 microbiology
Microbiology
law.invention
Matrix (chemical analysis)
03 medical and health sciences
chemistry.chemical_compound
law
Molecular Biology
Filtration
Eucalyptus
Chromatography
Bacteria
biology
Chemistry
Extraction (chemistry)
biology.organism_classification
Wood
DNA extraction
Woodchips
Metagenomics
Stress, Mechanical
DNA
Subjects
Details
- ISSN :
- 01677012
- Volume :
- 148
- Database :
- OpenAIRE
- Journal :
- Journal of Microbiological Methods
- Accession number :
- edsair.doi.dedup.....d6b6e18428af1b97f1d19ff0f5dec62e
- Full Text :
- https://doi.org/10.1016/j.mimet.2018.04.009