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Inclusion of 2-Mercaptoethanol in Lysis Buffer Could Interfere with Isolation of High Molecular Weight DNA from Freshwater Microalgae.

ABSTRACT: 2-mercaptoethanol (2-ME), alongside polyvinylpyrrolidone is commonly used in plant DNA extractions to deal with polyphenols, which could interfere with extraction and downstream applications. 2-ME is also commonly used to denature proteins and nucleases, especially RNAses. On the contrary, we found that the presence of 2-ME in lysis buffer interfered with DNA extraction from 12 strains of freshwater microalgae, resulting in DNA with poor integrity. We also found that the TNES-urea buffer, commonly used for preservation and DNA extraction from fish, appears as effective as the SDS and CTAB buffer for some microalgae strains. Results from our study suggests that the inclusion of 2-ME in DNA extraction protocols may be detrimental for isolation of good quality DNA from freshwater microalgae, and therefore recommend eliminating it or testing varying concentrations of 2-ME when developing species-specific extraction protocols for microalgae.

SUBMITTER: Yee W

PROVIDER: S-EPMC5801187 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Inclusion of 2-Mercaptoethanol in Lysis Buffer Could Interfere with Isolation of High Molecular Weight DNA from Freshwater Microalgae.

Yee Willy W Kumar Janushree Nanda JN Muthusamy Priya Devi PD

Indian journal of microbiology 20171214 1

2-mercaptoethanol (2-ME), alongside polyvinylpyrrolidone is commonly used in plant DNA extractions to deal with polyphenols, which could interfere with extraction and downstream applications. 2-ME is also commonly used to denature proteins and nucleases, especially RNAses. On the contrary, we found that the presence of 2-ME in lysis buffer interfered with DNA extraction from 12 strains of freshwater microalgae, resulting in DNA with poor integrity. We also found that the TNES-urea buffer, common ...[more]

PMID: 29434405

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Project description:Algae with potential biotechnological applications in different industries are commonly isolated from the environment in order to obtain pure (axenic) stocks that can be safely stored for long periods of time. To obtain axenic cultures, antibiotics are frequently employed, and cryopreservation is applied to preserve standing stocks. However, many of these now standard methods were developed using strains derived from pristine to near-pristine environments and cold to temperate regions. The potential effect of the said methods on the life cycle and biochemical profile of algae isolates from hyper-eutrophic and constant high-temperature tropical regions is not well understood. These effects could potentially render them unsuitable for their intended biotechnological application. In this study, we conducted a genetic characterization (18S rRNA) and evaluated the effect of purification (the use of the antibiotic chloramphenicol, CAP) and cryopreservation (dimethyl sulfoxide; DMSO-sucrose mix and glycerol) on the growth rate and lipid content of three new tropical freshwater algal isolates: Chorella sp. M2, Chlorella sp. M6, and Scenedesmus sp. R3, obtained from the Ecuadorian coast. The genetic and morphological characterization revealed a clear discrimination between these strains. All strains cultured with CAP exhibited a lower growth rate. Subsequent to cryopreservation, Chorella sp. M2, Chlorella sp. M6, and Scenedesmus sp. R3 presented no significant difference in growth rate between the cryopreservants. Further, a significantly higher lipid content was observed in the biomass cryopreserved with glycerol in relation to the DMSO-sucrose, with Chorella sp. M2 and Chlorella sp. M6 having twice as much as they had in the first treatment. These results highlight the relevance of selecting an appropriate method for storage, as the materials used can affect the biological performance of different tropical species, although it is still to be determined if the effects observed in this study are long lasting in subsequent cultures of these algae.

Project description:BackgroundRecent studies have demonstrated that microalga has been widely regarded as one of the most promising raw materials of biofuels. However, lack of an economical, efficient and convenient method to harvest microalgae is a bottleneck to boost their full-scale application. Many methods of harvesting microalgae, including mechanical, electrical, biological and chemical based, have been studied to overcome this hurdle.ResultsA new flocculation method induced by decreasing pH value of growth medium was developed for harvesting freshwater microalgae. The flocculation efficiencies were as high as 90% for Chlorococcum nivale, Chlorococcum ellipsoideum and Scenedesmus sp. with high biomass concentrations (>1g/L). The optimum flocculation efficiency was achieved at pH 4.0. The flocculation mechanism could be that the carboxylate ions of organic matters adhering on microalgal cells accepted protons when pH decreases and the negative charges were neutralized, resulting in disruption of the dispersing stability of cells and subsequent flocculation of cells. A linear correlation between biomass concentration and acid dosage was observed. Furthermore, viability of flocculated cells was determined by Evans Blue assay and few cells were found to be damaged with pH decrease. After neutralizing pH and adding nutrients to the flocculated medium, microalgae were proved to maintain a similar growth yield in the flocculated medium comparing with that in the fresh medium. The recycling of medium could contribute to the economical production from algae to biodiesel.ConclusionsThe study provided an economical, efficient and convenient method to harvest fresh microalgae. Advantages include capability of treating high cell biomass concentrations (>1g/L), excellent flocculation efficiencies (≥ 90%), operational simplicity, low cost and recycling of medium. It has shown the potential to overcome the hurdle of harvesting microalgae to promote full-scale application to biofuels from microalgae.

Dataset Information

Inclusion of 2-Mercaptoethanol in Lysis Buffer Could Interfere with Isolation of High Molecular Weight DNA from Freshwater Microalgae.

Publications

Inclusion of 2-Mercaptoethanol in Lysis Buffer Could Interfere with Isolation of High Molecular Weight DNA from Freshwater Microalgae.

Similar Datasets

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