Development of a SYBR safe™ technique for the sensitive detection of DNA in cesium chloride density gradients for stable isotope probing assays

Martineau, Christine; Whyte, Lyle G.; Greer, Charles W.

doi:10.1016/j.mimet.2008.01.016

Cited by 24 publications

(26 citation statements)

References 12 publications

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“…Separation of light and heavy DNA was performed as described previously (Martineau et al, 2008;Jones et al, 2011b) using 600 lL of the extracted DNA and 150 ng of spiked Escherichia coli DNA added to each ultracentrifuge tube (Singleton et al, 2005). After 40 h of ultracentrifugation at 175,800 g at 20°C in a TV-1665 vertical rotor (Sorvall), 250-lL fractions were collected through the bottom of the ultracentrifuge tubes via displacement from the top with water and a syringe pump, DNA purified and concentrated through ethanol precipitation, and the DNA from each fraction resuspended in 50 lL of TE buffer.…”

Section: Stable-isotope Probingmentioning

confidence: 99%

Biostimulation Reveals Functional Redundancy of Anthracene-Degrading Bacteria in Polycyclic Aromatic Hydrocarbon-Contaminated Soil

Singleton²,

Aitken³

2013

Environmental Engineering Science

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Stable-isotope probing was previously used to identify bacterial anthracene-degraders in untreated soil from a former manufactured gas plant site. However, subsequent pyrosequence analyses of total bacterial communities and quantification of 16S rRNA genes indicated that relative abundances of the predominant anthracene-degrading bacteria (designated Anthracene Group 1) diminished as a result of biological treatment conditions in lab-scale, aerobic bioreactors. This study identified Alphaproteobacterial anthracene-degrading bacteria in bioreactor-treated soil which were dissimilar to those previously identified. The largest group of sequences was from the Alterythrobacter genus while other groups of sequences were associated with bacteria within the order Rhizobiales and the genus Bradyrhizobium. Conditions in the bioreactor enriched for organisms capable of degrading anthracene which were not the same as those identified as dominant degraders in the untreated soil. Further, these data suggest that identification of polycyclic aromatic hydrocarbon-degrading bacteria in contaminated but untreated soil may be a poor indicator of the most active degraders during biological treatment.

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Section: Stable-isotope Probingmentioning

confidence: 99%

Biostimulation Reveals Functional Redundancy of Anthracene-Degrading Bacteria in Polycyclic Aromatic Hydrocarbon-Contaminated Soil

Singleton²,

Aitken³

2013

Environmental Engineering Science

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“…Ethidium bromide is the most common stain used for nucleic acid detection on electrophoretic gels. This dye emits fluorescence when intercalated into DNA bases of nucleic acids and can interfere in both DNA and RNA synthesis (Singer et al, 1999;Ohta et al, 2001;Martineau et al, 2008;Haines et al, 2015). Ethidium bromide is classified as a strong mutagen and is genotoxic at the typical concentration for gel staining (Schagat and Hendricksen, 2013;Haines et al, 2015).…”

Section: Resultsmentioning

confidence: 99%

“…Ethidium bromide is classified as a strong mutagen and is genotoxic at the typical concentration for gel staining (Schagat and Hendricksen, 2013;Haines et al, 2015). Moreover, this stain requires special waste treatment, consequently increasing costs for laboratories (Martineau et al, 2008). Finally, ethidium bromide needs to be visualized under UV illumination, which can damage the DNA (Gründemann and Schömig, 1996;Martineau et al, 2008).…”

Section: Resultsmentioning

confidence: 99%

“…Moreover, this stain requires special waste treatment, consequently increasing costs for laboratories (Martineau et al, 2008). Finally, ethidium bromide needs to be visualized under UV illumination, which can damage the DNA (Gründemann and Schömig, 1996;Martineau et al, 2008).…”

Section: Resultsmentioning

confidence: 99%

“…DNA fragment staining is usually performed using ethidium bromide, which is known for its mutagenicity (Singer et al, 1999;Martineau et al, 2008). …”

Section: Introductionmentioning

confidence: 99%

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SYBR safe<sup>TM</sup> efficiently replaces ethidium bromide in Aspergillus fumigatus gene disruption

Canela¹,

Takami²,

Ferreira³

2017

Genet. Mol. Res.

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ABSTRACT. Invasive aspergillosis is a disease responsible for high mortality rates, caused mainly by Aspergillus fumigatus. The available drugs are limited and this disease continues to occur at an unacceptable frequency. Gene disruption is essential in the search for new drug targets. An efficient protocol for A. fumigatus gene disruption was described but it requires ethidium bromide, a genotoxic agent, for DNA staining. Therefore, the present study tested SYBR safe TM , a non-genotoxic DNA stain, in A. fumigatus gene disruption protocol. The chosen gene was cipC, which has already been disrupted successfully in our laboratory. A deletion cassette was constructed in Saccharomyces cerevisiae and used in A. fumigatus transformation. There was no statistical difference between the tested DNA stains. The success rate of S. cerevisiae transformation was 63.3% for ethidium bromide and 70% for SYBR safe TM . For A. fumigatus gene disruption, the success rate for ethidium bromide was 100 and 97% for SYBR safe TM . In conclusion, SYBR safe TM efficiently replaced ethidium bromide, making this dye a safe and efficient alternative for DNA staining in A. fumigatus gene disruption.

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Strategies of fluorescence staining for trace total ribonucleic acid analysis by capillary electrophoresis with argon ion laser‐induced fluorescence

2015

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In this work, five fluorescent dyes (SYTO-9, SYBR Green I, SYBR Green II, SYBR Safe, and SYBR Gold) were used as both on-column and precolumn stains for total RNA analysis by CE-LIF with Ar ion laser excitation. In the on-column RNA stain, the SYTO-9 provided the highest fluorescence intensity and the lowest detectable concentration, as low as 10 pg/μL, while the SYBR Green II and SYBR Gold were adsorbed on the poly(ethylene oxide) thus affected the separation efficiency. As a precolumn stain, SYBR Gold was the most sensitive among the five dyes due to the strong affinity between the dye and RNA molecules. As a result, a single-cell quantity of RNA (10-30 pg per cell) could be detected by CE-LIF with precolumn staining by SYBR Gold. Because of the great savings of fluorescent dye using precolumn stain (one button dye may use for one million stain), this method is the best strategy for RNA staining in terms of cost-effectiveness and sensitivity.

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Development of a SYBR safe™ technique for the sensitive detection of DNA in cesium chloride density gradients for stable isotope probing assays

Cited by 24 publications

References 12 publications

Biostimulation Reveals Functional Redundancy of Anthracene-Degrading Bacteria in Polycyclic Aromatic Hydrocarbon-Contaminated Soil

Biostimulation Reveals Functional Redundancy of Anthracene-Degrading Bacteria in Polycyclic Aromatic Hydrocarbon-Contaminated Soil

SYBR safe<sup>TM</sup> efficiently replaces ethidium bromide in Aspergillus fumigatus gene disruption

Strategies of fluorescence staining for trace total ribonucleic acid analysis by capillary electrophoresis with argon ion laser‐induced fluorescence

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