Estimation of the Growth Rate of Mixed Ruminal Bacteria from Short-term DNA Radiolabeling

Broudiscou, Laurent; Geissler, Hanno; Broudiscou, A. F.

doi:10.1006/anae.1998.0157

Cited by 6 publications

(5 citation statements)

References 20 publications

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“…There are few studies that report DNA yields per gram of digesta sample. One study reported a yield of 2530 g of community DNA per gram of rumen digesta, by grinding cells in the presence of liquid nitrogen and cetyltrimethylammonium bromide (CTAB) extraction (18) and a second method employing lysozyme plus freeze-thaw and phenol/chloroform extraction recovered 28.630 g of community DNA per milliliter of rumen fluid (6). The modifications described here do not add a significant amount of time compared to these other methods but improve DNA yields substantially.…”

Section: Resultsmentioning

confidence: 99%

“…The physicochemical nature of these types of samples can also make DNA extraction particularly difficult (5). A number of methods have been used to extract community DNA from rumen digesta and fecal samples, such as enzymatic lysis and freeze-thaw (3,6), QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA, USA) (5,710), FastDNA SPIN Kit (Qbiogene, Carlsbad, CA, USA) (1113), bead beating (2,1417), and liquid nitrogen grinding (18). These methods support the recovery of DNA in a form that is suitable for use in various PCR-based studies of microbial diversity.…”

Section: Introductionmentioning

confidence: 99%

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Improved extraction of PCR-quality community DNA from digesta and fecal samples

2004

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Several DNA extraction methods have been reported for use with digesta or fecal samples, but problems are often encountered in terms of relatively low DNA yields and/or recovering DNA free of inhibitory substances. Here we report a modified method to extract PCR-quality microbial community DNA from these types of samples, which employs bead beating in the presence of high concentrations of sodium dodecyl sulfate (SDS), salt, and EDTA, and with subsequent DNA purification by QIA columns [referred to as repeated bead beating plus column (RBB+C) method]. The RBB+C method resulted in a 1.5- to 6-fold increase in DNA yield when compared to three other widely used methods. The community DNA prepared with the RBB+C method was also free of inhibitory substances and resulted in improved denaturing gradient gel electrophoresis (DGGE) profiles, which is indicative of a more complete lysis and representation of microbial diversity present in such samples.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Improved extraction of PCR-quality community DNA from digesta and fecal samples

2004

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“…DNA was extracted following the freeze-thaw and lysozyme-SDS lysis method [19], with slight modification [20]. To improve the DNA extraction from the biofilm samples, pre-lysis washing was conducted [21].…”

Section: Dna Extractionmentioning

confidence: 99%

Response of Planktonic and Benthic Microbial Community to Urban Pollution from Sewage Discharge in Jilin Reach of the Second Songhua River, China

Lin

Wang

Lin

et al. 2014

CLEAN Soil Air Water

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To investigate the role of the planktonic/benthic bacterial community in sewage polluted water, the biomass, suspended solids (SS), and chemical oxygen demand (COD) were measured downstream from a discharge point (P0) and reference water point (U1) in the Second Songhua River, China. In addition, 16S rDNA community was analyzed using MOTHUR and the bacterial abundance was assayed by real-time quantitative PCR. The results showed that the SS (14 mg L ). The bacterial diversity of polluted water had a lower richness than that of sediment at P0. Approximately 80% of the detected planktonic and benthic bacteria were anaerobic/ facultative anaerobic bacteria at P0. The most abundance of planktonic bacteria (10 8 copies ng À1) was observed at 100 m downstream of P0 where more ecological nutrients (COD, 64.07 mg L À1) accumulated. However, the benthic bacterial abundance reached the highest value 50 m downstream of P0 where the COD were not the highest. The relationship between the density of planktonic/benthic microbes and COD suggested that planktonic/benthic microbes possessed high degradation capabilities in river and provided good potential purification for the organic materials discharged from urban sewage.

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“…DNA was extracted following the "freeze-thaw and lysozyme-SDS lysis" method described by Broudiscou et al [14], with slight modification [15,16]. In order to improve DNA extraction from the activated sludge, pre-lysis washing was used beforehand [17].…”

Section: Dna Extractionmentioning

confidence: 99%