Psp spin stool dna plus kit

Manufactured by Stratec

Sourced in Germany, Switzerland

The PSP Spin Stool DNA Plus Kit is a laboratory equipment designed for the extraction and purification of DNA from stool samples. It provides a standardized and efficient method for isolating high-quality DNA from fecal material.

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54 protocols using psp spin stool dna plus kit

Fecal Microbiome Profiling Protocol

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In brief, approximately 2.5 g of a fresh fecal sample was collected from each participant using a plastic tube prefilled with the stool DNA stabilizer including a measuring spoon for sample collection (PSP Spin Stool DNA Plus Kit; STRATEC Molecular, Berlin, Germany). Bacterial DNA from fecal samples was extracted according to the manufacturer's instructions (PSP Spin Stool DNA Plus Kit; STRATEC Molecular). DNA concentrations were assayed using a NanoDrop 2000 Bioanalyzer at 260 nm (Thermo Fisher Scientific Inc., Waltham, MA, USA). 16S rRNA genes of distinct regions (16S V4/16S, 515F: GTGCCAGCMGCCGCGGTAA; 806R: GGACTACHVGGGTWTCTAAT) were amplified using specific primers [14 (link)].
The sequences were analyzed and those with ≥97% similarity were classified into the same OTUs. The representative sequence for each OTU was screened out for further annotation. The abundance information on the OTUs was normalized using a standard sequence number, corresponding to each sample with the least number of sequences as previously described [9 (link)].

Shi T.T., Hua L., Wang H, & Xin Z. (2019). The Potential Link between Gut Microbiota and Serum TRAb in Chinese Patients with Severe and Active Graves' Orbitopathy. International Journal of Endocrinology, 2019, 9736968.

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Stool Sample Collection and DNA Extraction

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Stool sample collection and DNA isolation were performed in a previous study^{4 (link)}. Briefly, stool samples were collected by study subjects into collection tubes pre-filled with DNA stabilizer (PSP Spin Stool DNA Plus Kit, STRATEC Molecular) and stored in the refrigerator until transport (for up to 3 days). After receipt of samples, they were transferred to −80 °C. DNA from both baseline and follow-up samples were extracted with the PSP Spin Stool DNA Plus Kit (STRATEC Molecular). Each extraction batch included one blank sample to assess potential contamination. (Of note, to prevent potential technical differences, DNA from baseline samples were extracted at the baseline^{5 (link)} and at follow-up^{4 (link)}, thus the baseline samples were thawed twice.)

van Kessel S.P., Auvinen P., Scheperjans F, & El Aidy S. (2021). Gut bacterial tyrosine decarboxylase associates with clinical variables in a longitudinal cohort study of Parkinsons disease. NPJ Parkinson's Disease, 7, 115.

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Fecal DNA Extraction from Difficult Bacteria

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Genomic DNA from fecal samples were isolated using the Stratec PSP Spin Stool DNA Plus kit using the difficult to lyse bacteria protocol from the manufacturer (STRATEC Molecular GmbH, Berlin, Germany). Each sample DNA was eluted into 100 μL of elution buffer provided by the Stratec PSP Spin Stool DNA Plus kit.

Morrison K.E., Jašarević E., Howard C.D, & Bale T.L. (2020). It's the fiber, not the fat: significant effects of dietary challenge on the gut microbiome. Microbiome, 8, 15.

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Microbial Diversity Profiling of Fecal, Vaginal, and Colonic Samples

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Fecal, vaginal fluid, and colonic samples were rapidly frozen in liquid nitrogen and subsequently stored at −80 °C before DNA extraction. Genomic DNA from fecal, vaginal and colon samples was isolated using the Stratec PSP Spin Stool DNA Plus kit using the difficult to lyse bacteria protocol from the manufacturer (STRATEC Molecular GmbH, Berlin, Germany). For each set of extractions, one blank swab exposed to laboratory air was processed as a negative control. The V4 region of the 16S rRNA gene was amplified using barcoded primers for the Illumina platform as previously described87 (link). Sequencing was performed on a MiSeq instrument (Illumina, San Diego, CA) using 250 base paired-end chemistry at the University of Pennsylvania Next Generation Sequencing Core. Mock communities, consisting of genomic DNA from 12 ATCC strains, were included as a positive control and for run-to-run quality control. Quality filtering and chimera checking yielded 15,471,769 quality-filtered sequences with a mean ± SD depth of 24,326 ± 4,1875 sequences per sample.

Jašarević E., Howard C.D., Misic A.M., Beiting D.P, & Bale T.L. (2017). Stress during pregnancy alters temporal and spatial dynamics of the maternal and offspring microbiome in a sex-specific manner. Scientific Reports, 7, 44182.

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Fecal Microbiome Sampling Protocol

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Weekly fecal samples were collected from 1 male and 1 female per litter per group from C57BL/6J cohort from postnatal day 21 to 63. Genomic DNA from fecal samples were isolated using the Stratec PSP Spin Stool DNA Plus kit as per the manufacturer’s protocol for “difficult-to-lyse bacteria” (STRATEC Molecular GmbH, Berlin, Germany).

Hill E.M., Howard C.D., Bale T.L, & Jašarević E. (2021). Perinatal exposure to tetracycline contributes to lasting developmental effects on offspring. Animal Microbiome, 3, 37.

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Quantifying Vaginal Microbiome Composition

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Genomic DNA from vaginal fluid recovered in 50 µL saline were isolated using the Stratec PSP Spin Stool DNA Plus kit using the difficult to lyse bacteria protocol from the manufacturer (STRATEC Molecular GmbH, Berlin, Germany). Each sample DNA was eluted into 100 µL of Elution Buffer provided by the Stratec PSP Spin Stool DNA Plus kit. Gene copy number of G. vaginalis rpoB was determined by quantitative real-time PCR analysis, performed on an Applied Biosystems QuantStudio6 Flex Real Time PCR System (Life Technologies, Grand Island, NY). Genomic DNA was extracted using Stratec PSP Spin Stool DNA Plus kit from vaginal swabs, TaqMan Assays specific for G. vaginalis rpoB, panbacterial 16 S rRNA genes, TaqMan Vaginal Microbiota Amplification Control, and TaqMan Fast Advanced Master Mix (Life Technologies). Gene copy number counts were calculated using a standard curve method and the 16 S rRNA gene level as an internal standard.

Jašarević E., Hill E.M., Kane P.J., Rutt L., Gyles T., Folts L., Rock K.D., Howard C.D., Morrison K.E., Ravel J, & Bale T.L. (2021). The composition of human vaginal microbiota transferred at birth affects offspring health in a mouse model. Nature Communications, 12, 6289.

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Shotgun Metagenomic Sequencing of Stool Microbiome

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Microbiome analysis was completed by the CHOP Microbiome Center. Bacterial DNA was isolated from approximately 200 mg of stool. The PSP® Spin Stool DNA Plus Kit (STRATEC Molecular GmbH, Berlin, Germany) was used and isolated DNA was quantified using the Quant-iT™ PicoGreen™ dsDNA Assay Kit (Invitrogen™, Eugene, Ore.). Genomic DNA was prepared for shotgun metagenomic sequencing using the Nextera® XT DNA Library Preparation Kit (Illumina®, San Diego, Calif.). DNA extraction blanks and DNA-free water were included as negative control samples to assess environmental and reagent contamination. Laboratory-generated mock communities consisting of DNA from Vibrio campbellii, Cryptococcus diffluens and lambda phage were included as positive control samples. DNA sequencing was carried out on an Illumina® HiSeq 2500 instrument, generating 125bp paired-end sequence reads. Reads were quality-filtered and trimmed to remove adapter sequences using Trimmomatic v. 0.36.^{17 (link)} Reads aligning to the human host genome (version hg38) were removed using BWA v. 0.7.17-r1188.^{18 (link)} Taxonomic annotations were generated by Kraken v. 1.0^{19 (link)} using the standard database with all complete bacterial, archaeal, and viral genomes in NCBI RefSeq.

Vajravelu M.E., Lee J.J., Mitteer L., Zemel B.S., Bittinger K, & De León D.D. (2021). Gut Microbiome Profile After Pancreatectomy in Infants With Congenital Hyperinsulinism. Pancreas, 50(1), 89-92.

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Metagenomic Shotgun Sequencing of Stool Samples

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Stool samples were brought chilled to the study center within 16 hours after fecal output where they were immediately frozen at −20 °C. Metagenomic DNA was isolated from up to 200 mg of stool sample using the PSP^® Spin Stool DNA Plus kit (Stratec Biomedical AG, Beringen, Switzerland) according to the manufacturer’s protocol with an integrated RNA digestion step using 100 mg/ml RNase A (Qiagen, Hombrechtikon, Switzerland). The DNA was brought to the Next Generation Sequencing Platform of the University of Bern, Switzerland, for metagenomic shotgun sequencing. The TruSeq DNA PCR-Free Library Preparation kit was used for library preparation for sequencing following standard pipelines of the Illumina HiSeq 3000 platform with up to ten samples pooled in one lane. The resulting 150 bp paired-end reads were quality filtered with Trimmomatic v.0.32^{43 (link)}. To remove sequences of human origin, all reads were mapped to the human reference genome hg19 using Bowtie2 v.2.2.4^{44 (link)} and only the unmapped reads were used for further analysis.

Zysset-Burri D.C., Keller I., Berger L.E., Neyer P.J., Steuer C., Wolf S, & Zinkernagel M.S. (2019). Retinal artery occlusion is associated with compositional and functional shifts in the gut microbiome and altered trimethylamine-N-oxide levels. Scientific Reports, 9, 15303.

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Microbiome Profiling with 16S rRNA Sequencing

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The protocol for the microbiome measurements was explained in detail earlier [8 (link)]. Briefly, this process included three steps: sample processing, DNA extraction and DNA sequencing. Sample processing: Participants were provided a stool collection kit (Commode Specimen Collection System, Fisher Scientific, Pittsburgh, PA, United States) to collect a stool sample within 24 h prior to their study visit. Aliquots were made in 36 h from the time of sample collection and stored at −80°C. DNA extraction: DNA isolation was performed with the use of the PSP Spin Stool DNA Plus Kit (Stratec, Germany). Barcoded primers (Eurofins Genomics, Louisville, KY, United States) were used to amplify the 16S rRNA gene section. DNA sequencing: After being cleaned (MinElute PCR Purification kit, Qiagen, Germantown, MD, United States), DNA libraries were pooled and subsequently sequenced on the MiSeq platform, 300 bp paired-end reads, at an average depth of 158,000 reads/sample (Illumina Inc., San Diego, CA, United States) in two batches; at the University of Pennsylvania Next-Generation Sequencing Center (UPenn NGSC, N = 107) and the Vanderbilt University Technologies for Advanced Genomics (VANTAGE) Core (N = 29).

Bagheri M., Shah R.D., Mosley J.D, & Ferguson J.F. (2021). A metabolome and microbiome wide association study of healthy eating index points to the mechanisms linking dietary pattern and metabolic status. European journal of nutrition, 60(8), 4413-4427.

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Multispecimen Microbiome and Transcriptome Isolation

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Microbial DNA was isolated from oral washes, induced sputum, and background controls (one specimen-control pair selected for every 10 patients) using the Purelink DNA Microbiome Kit (Invitrogen, Carlsbad, USA) and from stool using the PSP Spin Stool DNA Plus Kit (Stratec Biomedical, Birkenfeld, Germany). Human RNA was isolated from blood using the Tempus Spin RNA Isolation kit (Applied Biosystems). Contaminating genomic DNA was removed using the TURBO DNA-free kit (Ambion, Austin, USA), globin mRNA depleted using the GLOBINclear kit (Ambion, Austin, USA), RNA concentrated using the RNeasy MinElute Cleanup kit (QIAGEN, Hilden, Germany) and its quality assessed using a Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA).

Naidoo C.C., Nyawo G.R., Sulaiman I., Wu B.G., Turner C.T., Bu K., Palmer Z., Li Y., Reeve B.W., Moodley S., Jackson J.G., Limberis J., Diacon A.H., van Helden P.D., Clemente J.C., Warren R.M., Noursadeghi M., Segal L.N, & Theron G. (2021). Anaerobe-enriched gut microbiota predicts pro-inflammatory responses in pulmonary tuberculosis. EBioMedicine, 67, 103374.

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