Application of GelGreen™ in Cesium Chloride Density Gradients for DNA-Stable Isotope Probing Experiments

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 (13)C-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.