The frozen glycerol stocks from each culture medium were resuspended in GAM broth, and 100 µl of 10–5 to 10–8 dilutions were plated on the original selected medium to obtain a single colony (suspensions of glycerol stock obtained from GAM-medium growth were spread on GAM agar plates). Based on the 16S rRNA gene metagenomic analysis data, we isolated up to 200 colonies from each medium, and single colonies were streaked on fresh agar plates for replication. Genomic DNA was purified using the ZR-96 Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA, USA). The V3-V4 regions of the 16S rRNA gene were amplified using the 341F (5ʹ-CCTACGGGAGGCAGCAG-3ʹ) and 926R (5ʹ-CCGTCAATTCCTTTRAGTTT-3ʹ) primers. Purified PCR products were subjected to Sanger sequencing by a 3730xl DNA Analyzer (Applied Biosystems, Carlsbad, CA, USA). Taxonomic assignments of isolates were made with NCBI BLAST using the 16S rRNA gene sequence database; we considered candidate strains with < 97% nucleotide identity as novel bacterial species. For those candidates, single colonies from the replica plates were streaked onto fresh plates, and colonies were frozen at final concentration of 20% glycerol and 5% skim milk at −80°C.
Zr 96 zymoclean gel dna recovery kit
Manufactured by Zymo Research
Sourced in United States
The ZR-96 Zymoclean Gel DNA Recovery Kit is a laboratory equipment product designed for the efficient recovery of DNA fragments from agarose gel. The kit provides a simple and effective method for isolating DNA from gel slices, allowing for the purification and concentration of DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Miseq 2 250 v2 kit, by Illumina (2 mentions)
Hotstart readymix, by Roche (2 mentions)
3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Nextera xt dna sample preparation kit, by Illumina (1 mentions)
Phix control dna, by Illumina (1 mentions)
Qubit fluorometer 2, by Thermo Fisher Scientific (1 mentions)
Kapa hifi hotstart readymix, by Roche (1 mentions)
Low melt agarose gel, by National Diagnostics (1 mentions)
5 protocols using zr 96 zymoclean gel dna recovery kit
1
Isolation and Characterization of Novel Bacterial Strains
2
Bacterial 16S rRNA Gene Sequencing
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PCR was performed using primers for the variable 4 (V4) region of the bacterial 16S rRNA gene [20 (link)]. PCR reactions contained 12.5 ng DNA, 10 μM each primer, 12.5 μl 2× HotStart ReadyMix (KAPA Biosystems, Wilmington, MA, USA), and water to 25 μl. Cycling conditions were 95 °C for 3 min, then 25 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and finally 72 °C for 5 min. PCR products were purified by gel extraction from a 1% low-melt agarose gel using a ZR-96 Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA, USA). Individual samples were quantified by Qubit Fluorometer (Invitrogen, Carlsbad, CA, USA) and were equimolar pooled. The pool plus 5% PhiX control DNA was sequenced with the MiSeq 2 × 250 v2 kit (Illumina, San Diego, CA, USA) using custom sequencing primers [20 (link)]. All DNA sequences have been deposited in NCBI’s Short Read Archive (PRJNA393465).
3
Plasmid and Chromosome Sequencing
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Plasmid DNA was separated from chromosomal DNA by S1 nuclease-digestion followed by pulsed-field gel electrophoresis. Visible plasmid DNA and chromosomal DNA bands were extracted from the gel and purified using the ZR-96 Zymoclean gel DNA recovery kit (Zymo Research, Irvine, CA, United States). A DNA sequencing library was prepared using the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, United States) and was sequenced using an Illumina MiSeq and NextSeq 500 for plasmids and chromosomes, respectively. Sequencing reads (plasmid: 2 × 300-mer, 140 × median coverage; chromosome: 2 × 150-mer, 99 × median coverage) were assembled into contigs using the A5-MiSeq pipeline (Coil et al., 2015 (link)). Plasmid replicon typing was performed using the curated PlasmidFinder database at the CGE website1 (Carattoli et al., 2014 (link)).
4
Bacterial 16S rRNA Gene Amplification
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PCR was performed using universal primers flanking the V4 region of the bacterial 16S rRNA gene [13 (link)]. A total of 25 ng DNA, 0.4 μM each primer, 12.5 μl 2X KAPA HiFi HotStart ReadyMix (Kapa Biosystems, Wilmington, US), and water to 25 μl were used for one reaction per sample. Cycling conditions began with an initial denaturation of 95°C for 3 minutes followed by 25 cycles of 95 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 30 seconds, with a final extension at 72 °C for 5 minutes. PCR products were purified by gel extraction from a 1.0% low-melt agarose gel using the ZR-96 Zymoclean Gel DNA Recovery Kit (Zymo Research Corp, Irvine, US). Samples were quantified using a Qubit Fluorometer 2.0 (Invitrogen, Waltham, US) and pooled at equimolar concentrations. The pool plus 5% PhiX control DNA (Illumina, Inc, San Diego, US) was sequenced with the MiSeq 2x250 v2 reagent kit (Illumina, Inc, San Diego, US) using custom sequencing primers [13 (link)].
5
Bacterial 16S rRNA Sequencing Protocol
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PCR was performed using universal primers flanking the variable 4 (V4) region of the bacterial 16 S rRNA gene44 (link). A total of 50 ng DNA, 0.4 μM each primer, 12.5 μl 2X HotStart ReadyMix (KAPA Biosystems, Wilmington, MA, USA), and water to 25 μl were used for one reaction per sample. Cycling conditions were as follows: initial denaturation of 95 °C for 3 min followed by 25 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 5 min. PCR products were purified by gel extraction from a 1.0% low-melt agarose gel (National Diagnostics, Atlanta, GA) using a ZR-96 Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA). Samples were quantified by Qubit® Fluorometer and equimolar pooled. The pool plus 5% PhiX control DNA was sequenced with the MiSeq 2 × 250 v2 kit (Illumina, San Diego, CA, USA) using custom sequencing primers44 (link).
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