title: Application of GelGreen™ in Cesium Chloride Density Gradients for DNA-Stable Isotope Probing Experiments.
abstract: In this study, GelGreen™ was investigated as a replacement for SYBR® Safe to stain DNA in cesium chloride (CsCl) density gradients for DNA-stable isotope probing (SIP) experiments. Using environmental DNA, the usage of GelGreen™ was optimized for sensitivity compared to SYBR® Safe, its optimal concentration, detection limit for environmental DNA and its application in environmental DNA-SIP assay. Results showed that GelGreen™ was more sensitive than SYBR® Safe, while the optimal dosage (15X concentration) needed was approximately one-third of SYBR® Safe, suggesting that its sensitivity was three times more superior than SYBR® Safe. At these optimal parameters, the detection limit of GelGreen™-stained environmental DNA was as low as 0.2 μg, but the usage of 0.5 μg environmental DNA was recommended to produce a more consistent DNA band. In addition, a modified needle extraction procedure was developed to withdraw DNA effectively by fractionating CsCl density gradients into four or five fractions. The successful application of GelGreen™ staining with 13C-labeled DNA from enriched activated sludge suggests that this stain was an excellent alternative of SYBR® Safe in CsCl density gradients for DNA-SIP assays.
authors.affiliation: National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse Technology, College of Environmental and Energy Engineering, Beijing University of Technology, Beijing, China.
authors.forename: Jingfeng
authors.identifier: http://orcid.org/0000-0002-5430-7812
authors.initials: J
authors.lastname: Gao
authors.affiliation: National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse Technology, College of Environmental and Energy Engineering, Beijing University of Technology, Beijing, China.
authors.forename: Kailing
authors.initials: K
authors.lastname: Pan
authors.affiliation: National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse Technology, College of Environmental and Energy Engineering, Beijing University of Technology, Beijing, China.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Li
authors.affiliation: National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse Technology, College of Environmental and Energy Engineering, Beijing University of Technology, Beijing, China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Fan
authors.affiliation: National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse Technology, College of Environmental and Energy Engineering, Beijing University of Technology, Beijing, China.
authors.forename: Lixin
authors.initials: L
authors.lastname: Sun
authors.affiliation: Research and Development Center of Beijing Drainage Group Co., Ltd., Beijing, China.
authors.forename: Shujun
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Research and Development Center of Beijing Drainage Group Co., Ltd., Beijing, China.
authors.forename: Yongqing
authors.initials: Y
authors.lastname: Gao
mesh_terms: D002499:Centrifugation, Density Gradient / Q000379:methods*; D002586:Cesium / Q000737:chemistry*; D002712:Chlorides / Q000737:chemistry*; D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D018628:Gene Dosage / Q000235:genetics; D007553:Isotope Labeling / Q000379:methods; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics
chemical_list: D002712:Chlorides; D012336:RNA, Ribosomal, 16S; D002586:Cesium; D004247:DNA; C028019:cesium chloride