DNA was extracted from a total of 36 soil samples and 2 fallen leaves of E. polyandra using ISOIL (Nippon Gene Co., Ltd., Tokyo, Japan). The average amount of the soil samples used for DNA extraction was 0.30 g (s.d., 0.01 g), and that ofE. polyandra samples was 0.04 g and 0.06 g. The concentration of each extracted DNA sample was measured using a Qubit2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA).
Isoil
Manufactured by Nippon Gene
Sourced in Japan
ISOIL is a lab equipment product designed for the isolation and purification of nucleic acids. It functions as a tool for extracting and purifying DNA or RNA from various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Isoil for beads beating kit, by Nippon Gene (1 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
35 μm nylon mesh, by Corning (1 mentions)
Isoplant 2 dna extraction kit, by Nippon Gene (1 mentions)
Pcr4 topo vector, by Thermo Fisher Scientific (1 mentions)
5 protocols using isoil
1
DNA Extraction from Soil and Leaf
2
Quantifying Soil and Root Microbiome
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Total soil DNA was extracted from 500 mg of each soil sample using ISOIL for Beads Beating kit (Nippon Gene Co., Ltd., Tokyo, Japan) following the protocol of the manufacturer. For root DNA extraction, root tissue up to 1 cm from the root tip was separated from root sample using a sterile pair of scissors after washing the roots three times with sterile deionized water in order to remove adhering soil particles. Total root DNA was extracted from the 30 roots tips with two laboratory replicates using ISOIL (Nippon Gene Co., Ltd., Tokyo, Japan) without bead beating to avoid extracting root DNA (Toju and Sato, 2018 (link)). The DNA was eluted in 100 μl of TE buffer.
Bacterial and fungal gene copy numbers were quantified by real-time SYBR Green PCR assays in a StepOne Real-Time PCR System (Life Technologies Japan, Tokyo, Japan) with the 16S rRNA gene primer pair Eub338 and Eub518 and the 18S rRNA gene primer pair 5.8 s and ITS1f (Rousk et al., 2010 (link)) as previously described (Sawada et al., 2021 (link)). Standard curves were obtained using a 10-fold serial dilution of a plasmid containing either the Escherichia coli 16S rRNA gene or the Saccharomyces cerevisiae 18S rRNA gene. The measurements were expressed as copy numbers on an oven-dry soil weight basis (105°C, 24 h).
Bacterial and fungal gene copy numbers were quantified by real-time SYBR Green PCR assays in a StepOne Real-Time PCR System (Life Technologies Japan, Tokyo, Japan) with the 16S rRNA gene primer pair Eub338 and Eub518 and the 18S rRNA gene primer pair 5.8 s and ITS1f (Rousk et al., 2010 (link)) as previously described (Sawada et al., 2021 (link)). Standard curves were obtained using a 10-fold serial dilution of a plasmid containing either the Escherichia coli 16S rRNA gene or the Saccharomyces cerevisiae 18S rRNA gene. The measurements were expressed as copy numbers on an oven-dry soil weight basis (105°C, 24 h).
3
Biofilm Profiling in Circulating Water Pipes
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Biofilms on the pipes of the experimental model of the circulating water pipe were collected 10 days after the start of the circulation cycle. DNA was extracted from the biofilms using ISOIL (Nippon Gene, Tokyo, Japan) according to the instructions of the manufacturer. The V4 region of the prokaryotic 16S rRNA was amplified using 515F/806R primers (Caporaso et al., 2011) . Amplification products with six replicates per sample were sequenced using the MiSeq instrument and MiSeq reagent kit v2 (300 cycles) (Illumina, San Diego, CA, USA) by Bioengineering Lab. Co., Ltd. (Kanagawa, Japan) .
4
Soil Bacterial Metagenomic DNA Extraction
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Soil samples were collected beneath zelkova trees (Shinjuku-ku, Tokyo, Japan). Soil metagenomic DNA was isolated and purified from 1.5 g of soil sample by using ISOIL (Nippon Gene; Tokyo, Japan) according to the manufacturer’s protocol. For isolating bacterial fractions, 1.5 g of soil samples were suspended to 3 mL of DPBS(−). After vortexing thoroughly, the mixture was left to stand for 5 min. We collected the supernatant and centrifuged at 200 × g for 5 min. The supernatant was collected and filtered through a cell-strainer cap with a 35-μm nylon mesh (Corning, NY, USA). After the filtration, the cell suspension was centrifuged at 13,000 × g for 20 min. The pellet was washed twice with DPBS(−) before droplets generation.
5
Methanogenic Archaea Isolation and Identification
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Total DNA was extracted from the enrichment cultures and isolates using an ISOIL and ISOPLANT II DNA extraction kit (NIPPON GENE, Tokyo, Japan), respectively, and according to the manufacturer’s protocol. The archaeal and bacterial 16S rRNA genes were amplified from enrichment cultures using the primer pairs Arc21F and Univ1490R and Bac8F and Bac1492R, respectively. The products were purified using a MonoFas DNA purification kit, cloned into the pCR4-TOPO vector (Thermo Fisher Scientific) and sequenced using the dideoxynucleotide chain termination method with BigDye terminator reagents (Thermo Fisher Scientific).
The 16S rRNA and mcrA gene sequences of methanogenic isolates were determined. The ME3MFe and ME2r’ primer pair was used for mcrA gene amplification. The bacterial 16S rRNA gene was amplified using the primer pair Bac8F and Bac1492R to check the purity of the methanogenic isolate.
The 16S rRNA and mcrA gene sequences of methanogenic isolates were determined. The ME3MFe and ME2r’ primer pair was used for mcrA gene amplification. The bacterial 16S rRNA gene was amplified using the primer pair Bac8F and Bac1492R to check the purity of the methanogenic isolate.
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