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Spri beads

Manufactured by GE Healthcare
Sourced in United States

SPRI beads are paramagnetic beads used for the purification and isolation of nucleic acids, such as DNA and RNA, from various biological samples. These beads utilize a solid-phase reversible immobilization (SPRI) technology, allowing for efficient capture, washing, and elution of the target molecules. SPRI beads provide a simple and scalable solution for nucleic acid purification in a range of applications, including next-generation sequencing, PCR, and other molecular biology workflows.

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4 protocols using spri beads

1

16S rRNA Gene Amplicon Sequencing

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Libraries were generated using barcoded PCR primers 27F 5′-AGAGTTTGATCCTGGCTCAG-3′ and 338R 5′-TGCTGCCTCCCGTAGGAGT-3′ annealing to the V1–V2 region of the 16S rRNA gene36 (link). PCR reactions were carried out in quadruplicate using Q5 High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA). After amplification, quadruplicate PCR reactions were pooled and then purified using a 1:1 volume of SPRI beads (GE HealthCare, Chicago, IL). DNA in each sample was then quantified using Quant-iT PicoGreen Assay Kit (Thermo Fisher) and pooled in equal molar amounts. The resulting library was sequenced on the MiSeq instrument (Illumina, San Diego, CA) using 2 × 250 bp chemistry. Extraction blanks and DNA-free water were subjected to the same amplification and purification procedure to allow for empirical assessment of environmental and reagent contamination. Positive controls, consisting of eight artificial 16S gene fragments synthesized in gene blocks and combined in known abundances, were also included37 (link).
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2

Amplicon sequencing of gut microbiome

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Mucosal-associated DNA from the three gut sites was processed using barcoded PCR primers annealing to the V1-V2 region of the 16S rRNA gene. PCR reactions were carried out in quadruplicate using Q5 High-Fidelity DNA Polymerase (NEB, Ipswich, MA). Each PCR reaction contained 0.5 uM of each primer, 0.34 U Q5 Pol, 1X Buffer, 0.2 mM dNTPs, and 5.0 ul DNA in a total volume of 25 ul. Cycling conditions were as follows: 1 cycle of 98C for 1 m; 30 cycles of 98C for 10 s, 56 C for 20 S, and 72C for 20 sec; 1 cycle of 72C for 8 m. Replicate reactions were combined, cleaned using SPRI beads (GE Healthcare Life Sciences), and quantified using PicoGreen (Thermo Fisher Scientific). An equimolar amount of each sample was pooled, and the resulting library was sequenced on the Illumina MiSeq using 2×250 bp chemistry. Extraction blanks and DNA free water were processed in parallel to allow for empirical assessment of environmental and reagent contamination. Positive controls, consisting of eight artificial 16S gene fragments, were also included (Integrated DNA Technologies).
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3

16S rRNA Gene Amplification and Sequencing

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Bacterial DNA was extracted as described previously [43 (link)] using the PureLink kit (K182002, Invitrogen) and quantified using the Quant-iT PicoGreen Assay Kit (P7589, Invitrogen). The 16S ribosomal RNA genes were amplified using bar-coded PCR primers targeted to the V1-V3 hypervariable region (FP 5′-AGAGTTTGATCCTGGCTCAG-3′; RC 5-ATTACCGCGGCTGCTGG-3′). PCR reactions were carried out in quadruplicate using Accuprime Taq HiFi (12346086, Invitrogen). Each PCR reaction contained 0.2 µM of each primer, 1 U Accuprime Taq HiFi, 1× Buffer II, and 2 µL DNA in a total volume of 25 µL. Cycling conditions are as follows: 1 cycle of 95 °C for 2 min; 32 cycles of 95 °C for 20 s, 60 °C for 30 s, and 72 °C for 60 s; 1 cycle of 72 °C for 5 min. The resulting 16S rDNA amplicons were purified using a 1:1 volume of SPRI beads (09-981-123, GE Healthcare, Chicago, IL, USA), quantified using PicoGreen, pooled in equal amounts, and sequenced on the Illumina MiSeq using 2 × 300 bp chemistry. Extraction blanks and DNA-free water were subjected to the same amplification and purification procedure to assess potential environmental contamination. Library preparation and sequencing were performed at the CHOP Microbiome Center (University of Pennsylvania, Philadelphia, PA, USA).
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4

16S rRNA Gene Amplicon Sequencing from Gut Mucosal Samples

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Mucosal-associated DNA from the three gut sites was processed using barcoded PCR primers annealing to the V1-V2 region of the 16S rRNA gene. PCR reactions were carried out in quadruplicate using Q5 High-Fidelity DNA Polymerase (NEB, Ipswich, MA). Each PCR reaction contained 0.5 μM of each primer, 0.34 U Q5 Pol, 1X Buffer, 0.2 mM dNTPs, and 5.0 μl DNA in a total volume of 25 μl. Cycling conditions were as follows: 1 cycle of 98 °C for 1 m; 30 cycles of 98 °C for 10 s, 56 °C for 20 s, and 72 °C for 20 s; 1 cycle of 72 °C for 8 min. Replicate reactions were combined, cleaned using SPRI beads (GE Healthcare Life Sciences), and quantified using PicoGreen (Thermo Fisher Scientific). An equimolar amount of each sample was pooled, and the resulting library was sequenced on the Illumina MiSeq using 2 × 250 bp chemistry. Extraction blanks and DNA free water were processed in parallel to allow for empirical assessment of environmental and reagent contamination. Positive controls, consisting of eight artificial 16S gene fragments, were also included (Integrated DNA Technologies).
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