Approximately 25 mg aliquots of stool were weighed, and then DNA purified using the UltraClean Fecal DNA kit (MoBio Laboratories, Inc., Carlsbad, CA). 16S rRNA gene copy numbers were measured in duplicate for each sample using a panbacterial quantitative PCR TaqMan assay [87 (link)]. 16S copy numbers were estimated by reference to a dilution series standard curve of a plasmid carrying a 16S rRNA gene [88 (link)]. Results were averaged for technical replicates and normalized by the weight of each stool aliquot. Log10-transformed data were analyzed across all four timepoints by ANOVA and pairs of timepoints by Tukey Honest Significant Difference tests.
Ultraclean fecal dna kit
Manufactured by Qiagen
The UltraClean Fecal DNA Kit is a laboratory equipment product designed for the extraction and purification of DNA from fecal samples. It provides a reliable and efficient method for obtaining high-quality DNA from various types of fecal material.
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Lab products found in correlation
5 protocols using ultraclean fecal dna kit
1
Quantitative Analysis of Gut Microbiome
2
Fecal Microbiome Profiling in Mice
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For composition analyses, total DNA was extracted from fecal pellets collected from untreated, phytonutrient or antibiotic treated mice using the Ultra Clean Fecal DNA kit (Mo Bio Laboratories, Carlsbad, CA), including physical disruption using a FastPrep instrument (MP Biomedicals, Solon, OH) for 60 sec at level 5. 16S rRNA gene fragments were PCR amplified with nucleotide-bar-coded primer pairs 27F: 5′-AGAGTTTGATCMTGGCTCAG-3′and 510R: 5′-GWATTACCGCGGCKGCTG-3′. PCR products were gel-purified (QIAquick gel extraction kit, Qiagen, Valencia, CA). Each amplicon (100 ng) was pooled and pyrosequenced using a 454 Titanium platform (Roche, Branford, CT).
3
Fecal Microbiome Profiling via 16S rRNA Sequencing
4
Fecal Microbiome Profiling via 16S rRNA Sequencing
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DNA was extracted from fecal pellets using the UltraClean Fecal DNA Kit (MO BIO Laboratories). Amplicons were generated using oligonucleotide primers that target approximately 300 bp of the V4 variable region of the 16S rRNA gene (primers 515F and 806R) 40 (link) and also were barcoded and pooled to construct the sequencing library, followed by sequencing with an Illumina MiSeq instrument to generate paired-end 150×150 reads. The software package QIIME 1.7.0 was used to analyze, display, and generate figures of microbiome data using a previously defined method41 (link). Bacterial DNA of Enterobacteriaceae and Bacteriodetes from fecal content was quantified using six-point standard curves constructed with reference bacteria specific for each bacterial group measured by qPCR in the 7500 Fast Sequence Detection System (Applied Biosystems). All the reactions were set up in Fast SYBR Green Master Mix (Applied Biosystems) at 20 µL total volume. Copy number of Enterobacteriaceae and Bacteriodetes was normalized to copy number of universal bacteria. The initialization step was 95°C for 10 minutes, the amplification step had 40 cycles of 95°C for 10 seconds followed by optimal annealing temperature for 45 seconds. The primers and reaction conditions are listed in Table S1 .
5
Fecal DNA Extraction Protocol
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Approximately 0.5-g aliquots of feces from each sample were washed twice with 5 mL of PBS (pH 7.4) and were subjected to DNA extraction using an Ultra-Clean Fecal DNA Kit (Mo Bio Laboratories, Carlsbad, CA). Approximately 100 ng of DNA was used for PCR amplification of the 16S rRNA gene in samples.
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