收录:
摘要:
In this study, GelGreen (TM) was investigated as a replacement for SYBR (R) Safe to stain DNA in cesium chloride (CsCI) density gradients for DNA-stable isotope probing (SIP) experiments. Using environmental DNA, the usage of GelGreen (TM) was optimized for sensitivity compared to SYBR (R) Safe, its optimal concentration, detection limit for environmental DNA and its application in environmental DNA-SIP assay. Results showed that GelGreen (TM) was more sensitive than SYBR (R) Safe, while the optimal dosage (15X concentration) needed was approximately one-third of SYBR (R) Safe, suggesting that its sensitivity was three times more superior than SYBR (R) Safe. At these optimal parameters, the detection limit of Gel-Green (TM)-stained environmental DNA was as low as 0.2 mu g, but the usage of 0.5 mu g 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 CsCI density gradients into four or five fractions. The successful application of Gel-Green (TM) staining with C-13-labeled DNA from enriched activated sludge suggests that this stain was an excellent alternative of SYBR (R) Safe in CsCI density gradients for DNA-SIP assays.
关键词:
通讯作者信息:
电子邮件地址: