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Abi prism 373 dna sequencer

Manufactured by Thermo Fisher Scientific
Sourced in United States

The ABI PRISM 373 DNA Sequencer is a laboratory instrument used for DNA sequencing. It utilizes capillary electrophoresis technology to separate and detect fluorescently labeled DNA fragments. The instrument can be used to determine the nucleotide sequence of DNA samples.

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4 protocols using abi prism 373 dna sequencer

1

Bacterial 16S rRNA Sequencing Protocol

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Antimicrobial-resistant isolates were identified using bacterial 16S rRNA gene sequence analysis. For amplification of the 16S rRNA genes, isolates were cultured in Tryptic soy broth (TSB2, BBL BD Becton Dickinson™) with 2% NaCl at 22 °C for 12–24 h and centrifuged at 9000× g for 3 min using an Eppendorf 5415D (Eppendorf AG, Hamburg, Germany) microcentrifuge to obtain a pellet. DNA extraction was carried out using the Wizard® Genomic DNA Purification commercial kit (Promega, Madison, WI, USA) following the supplier’s instructions, and the obtained DNA samples were stored at −20 °C until analysis.
The amplification of the 16S ribosomal genes of the isolates was carried out using PCR, following the methodology described by Opazo et al. [37 (link)]. The resulting amplified PCR products were sequenced with Macrogen (Rockville, MD, USA), using the ABI PRISM 373 DNA Sequencer (Applied Biosystems, Foster City, CA, USA). The sequences were edited and matched to the Ribosomal Database Project (http://rdp.cme.msu.edu/, accessed on 5 January 2024) to identify the bacterial isolates. The partial sequences of the 16S rDNA gene belonging to each isolate were deposited in the GenBank database under the accession numbers shown in Tables S1 and S2 (Supplementary Materials).
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2

Bacterial Identification via 16S rRNA Sequencing

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Isolates were cultured in Tryptic soy broth (Oxoid, Hants, UK) at 22 °C for 12–24 h and centrifuged at 9000 g for 3 min using an Eppendorf 5415D microcentrifuge to obtain a pellet. DNA extraction was carried out using the Wizard Genomic DNA Purification commercial kit (Promega, Madison, WI, USA) following the supplier’s instructions, and the obtained DNA samples were stored at −20 °C until analysis. The amplification of the 16S ribosomal genes of the isolates was carried out by PCR, following the methodology described by Opazo et al. [32 (link)]. The resulting amplified PCR products were sequenced by Macrogen (Rockville, MD, USA) using the ABI PRISM 373 DNA Sequencer (Applied Biosystems, Foster City, CA, USA). The sequences were edited and matched to the Ribosomal Database Project [33 ] to identify the bacterial isolates. Isolates exhibiting in their 16S rRNA gene sequence a similarity score of ≤99.4% with a nearest neighbor were not identified to the species level.
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3

PCR Product Purification and Sequencing

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Positive PCR products were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) and confirmed by sequencing with an ABI PRISM 373 DNA Sequencer (Applied Biosystems by Life Technologies Corporation, Monza, Italy). The results were analyzed using the Basic Local Alignment Search Tool (BLAST; www.blast.ncbi.nlm.nih.gov) and the sequence analyses were carried out using the National Center for Biotechnology Information GeneBank.
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4

Construction of HIV-1 LTR-based Luciferase Plasmids

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Construction of HIV-1 LTR-based luciferase expression plasmid: wild-type (WT CD12-luc) HIV-1 LTR-Luc (containing the HIV-1 LTR U3 and R regions), its 5′ truncated mutant CD23-Luc (containing positions -117 to +80 of HIV-1 LTR), CD52-Luc (containing positions -65 to +80 of HIV-1 LTR) and the NF-κB mutant HIV-1 LTR-Luc, where NF-κB sites were mutated in WT HIV-1 LTR-Luc, were prepared as previously described [26] [27] [28] . Sp1, AP-4 or LSF mutant HIV-1 LTR-Luc plasmids were generated by PCR using a QuikChange site-directed mutagenesis kit (Stratagene, Inc., La Jolla, CA, USA) as described previously [14, 26] . The following mutant sequences were utilized: Sp1 mutant HIV-1 LTR-Luc, TCG ttA; AP4 mutant HIV-1 LTR-Luc, CAG CTc; LSF mutant HIV-1 LTR-Luc, CAG tcG (consensus sequences of the binding sites are underlined and the mutated sequences are denoted by lower case letters). All constructs were confirmed by dideoxynucleotide sequencing using the ABI PRISM® dGTP BigDye™ Terminator v3.0 Ready Reaction Cycle Sequencing kit (PerkinElmer, Inc., Waltham, MA, USA) on an ABI PRISM® 373 DNA Sequencer (Applied Biosystems, Foster City, CA, USA). Sp1-Luc [29] was kindly provided by Dr. K. Kohno.
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