RNA was extracted from 200 μl of patient sample using the ThermoFisher MagMAX Viral/Pathogen II nucleic acid isolation kits with MagMAX magnetic beads and MS2 phage internal control, using the automated ThermoFisher Kingfisher flex magnetic particle processor (11 ).
Flex magnetic particle processor
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Flex Magnetic Particle Processor is a laboratory instrument designed for the automated processing of magnetic particles. It is used to separate, wash, and resuspend magnetic particles in a controlled and consistent manner.
Automatically generated - may contain errors
Lab products found in correlation
Q5 hot start high fidelity polymerase, by New England Biolabs (1 mentions)
Magmax viral pathogen nucleic acid isolation kit, by Thermo Fisher Scientific (1 mentions)
96 deep well head, by Thermo Fisher Scientific (1 mentions)
Magmax viral pathogen 2 mvp 2 nucleic acid isolation kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using flex magnetic particle processor
1
Automated Viral RNA Extraction
2
16S rRNA Gene Amplicon Sequencing
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DNA was extracted using a ZymoBIOMICS DNA Extraction kit on a KingFisher Flex Magnetic Particle Processor (Thermo Scientific). Mock communities (Zymo) and empty wells were included in the extraction plates as positive and negative controls, respectively. 16S-rRNA metabarcoding was conducted as described previously [42 (link)]. The V4 sub-region of the 16S SSU rRNA was amplified using 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT). Reactions were performed in 25 µl volumes using Q5 Hot Start High-Fidelity Polymerase (New England Biolabs) with 0.2 µM of each primer. Reaction conditions were 98 ℃ for 30 s, 30 cycles of 98 ℃ for 30 s, 50 ℃ for 30 s, and 72 ℃ for 2 min, and a final extension at 72 ℃ for 10 min. PCR products were normalized using a Just-A-Plate normalization kit (Charm Biotech). Amplicon pools were sequenced at the ASGPB Genomics Core at the University of Hawai’i at Manoa using Illumina MiSeq V3 600 chemistry (Illumina, San Diego, CA, USA).
3
Quantification of West Nile Virus RNA
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Vero and CT (Culex tarsalis derived) cells were infected with bcWNV and rWT-WNV at MOIs of 0.1 and 1.0, for 1 h, and washed three times with PBS before fresh growth media was added. Supernatant was sampled daily and RNA was extracted using the Omega MagBind Viral DNA/RNA 96 kit on the KingFisher Flex Magnetic Particle Processor (ThermoFisher). qRT-PCR was performed using One-Step SuperScript qRT-PCR kit (Invitrogen) and WNV envelope primer and probes (Table S2 ). RNA copies/ml was extrapolated using an RNA standard. The RNA standard was generated by amplifying ∼1 kb of sequence surrounding the envelope region of the WNV genome using a forward primer containing a T7 promoter sequence. The resulting amplicon was transcribed with T7 polymerase, and RNA was extracted and quantified.
4
Comprehensive SARS-CoV-2 Genome Sequencing
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WGS was performed using the Scripps PrimalSeq-Nextera XT protocol, based on the ARTIC protocol, with some modifications. Sequencing was performed on all available samples with adequate viral load (i.e., Ct value < 30) or upon special request by the infection control service related to cluster investigations as previously described10 (link),11 (link). Briefly, nucleic acids extraction was performed on the KingFisher Flex Magnetic Particle Processor using the MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher Scientific, Waltham, MA) with WGS performed as paired end (2 × 150 base pair read) on an Illumina Miseq (Illumina, San Diego, CA USA). Amplicon sequencing was performed using the most current version of the ARTIC primers (version 3 during the Alpha wave and the version 4 during the Omicron wave). The two versions of the ARTIC protocols differ in the sets of primers used and updated versions are used to address possible sequence dropout as the SARS-CoV-2 genome evolves. Primer sets were obtained from Integrated DNA Technologies (Coralville, IA). All whole genome sequences passing quality check were analyzed for lineage assignments using the DRAGEN COVID lineage app based on the Pangolin software (https://github.com/cov-lineages/pangolin ; version 4.1.2 pangolin-data 1.13).
5
Automated Magnetic Bead-Based Viral RNA Extraction
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The MagMAX viral/pathogen II (MVP II) nucleic acid isolation kit (Applied Biosystems) is a nucleic acid purification kit based on magnetic bead technology on the market. Extraction of viral nucleic acid was conducted on the KingFisher Flex Magnetic Particle Processor with a 96 Deep-Well Head (Thermo Scientific) according to kit instructions. Sample volumes were 200 µL, with elution volumes of 100 µL.
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