U. diversum ATCC 49782 and 45 isolates were provided by the Mycoplasma laboratory of the Institute of Biomedical Sciences - University of São Paulo (USP). Some strains were isolated from cows that had granulomatous vulvovaginitis, and others were isolated from the semen of healthy bulls. The isolates were obtained from four states: 19 isolated in São Paulo (farms 1, 2, 4, 8 and 9), 2 isolated in Mato Grosso do Sul (farm 3), 1 in Minas Gerais (farm 6), and 22 in Bahia (farms 10, 11, 12, 13). One milliliter of each sample previously-stored in UB medium was grown in 9 ml of the same medium at 37 °C for 24 to 48 h [7 (link)]. After growth, bacterial DNA was extracted using the NucleoSpin kit (Macherey-Nagel, Germany) following the manufacturer’s instructions. After growth, bacterial DNA was extracted using the NucleoSpin kit (Macherey-Nagel, Germany) following the manufacturer’s instructions.
Nucleospin kit
Manufactured by Macherey-Nagel
Sourced in Germany, United States, Switzerland, France
The NucleoSpin kit is a laboratory product designed for the purification of nucleic acids, such as DNA and RNA, from various biological samples. It employs silica-membrane technology to efficiently capture and purify the target molecules, while removing contaminants. The core function of the NucleoSpin kit is to provide a reliable and streamlined method for the extraction and isolation of high-quality nucleic acids for further analysis and applications.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (7 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (5 mentions)
Iscript cdna synthesis kit, by Bio-Rad (5 mentions)
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (5 mentions)
Sybr green pcr supermix, by Thermo Fisher Scientific (4 mentions)
Stepone real time pcr instrument, by Thermo Fisher Scientific (4 mentions)
Moloney murine leukemia virus reverse transcriptase, by Promega (4 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (4 mentions)
Lightcycler96 platform, by Roche (3 mentions)
Itaq universal probes one step rt qpcr kit, by Bio-Rad (3 mentions)
C1000 touch thermal cycler cfx96 instrument, by Bio-Rad (3 mentions)
Steponeplus apparatus, by Thermo Fisher Scientific (3 mentions)
Abi stepone software v2, by Thermo Fisher Scientific (3 mentions)
Qiamp viral rna kit, by Qiagen (3 mentions)
Cfx96 real time pcr system, by Bio-Rad (3 mentions)
Ssofast evagreen supermix kit, by Bio-Rad (3 mentions)
Nanodrop, by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
156 protocols using nucleospin kit
1
Isolation and Identification of U. diversum
2
Replacing pTight with TRE3G Promoter
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First, the 2nd generation pTight promoter from the RetroX-pTight::MCS_PGK::GpNLuc (Addgene plasmid # 70185) was replaced with a 3rd generation doxycycline response promoter; TRE3G. Briefly, the pTight promoter was excised using BamHI/XhoI restriction enzymes (New England Biosciences, cat#; R3136 and R0146, respectively). The BamHI digested vector was blunted using T4 polymerase (NEB, cat#; M0203) prior to the XhoI digestion in order to generate a blunt end/sticky end vector backbone. The dually digested plasmid was run in a 1% agarose gel and the digested backbone fragment (∼7150 bp) was excised and purified (Macherey-Nagel PCR clean up kit, cat. #: 740609). Next, the TRE3G promoter was excised from a previously published plasmid (TRE-KRAB-dCas9-IRES-BFP, Addgene plasmid # 85449) using SmaI/XhoI restriction enzymes (NEB, cat#; R0141). The digested vector backbone and insert, 1:6 ratio, were ligated overnight using T4 DNA ligase (NEB, cat#; M202) and transformed into homemade (Zymo Research, cat#; T3001) chemically competent E.coli Stable3 cells (NEB, cat#; C304). Multiple clones were miniprepped (Macherey-Nagel NucleoSpin kit, cat. #: 740588) and sequence verified (Eton Biosciences, Inc). All enzyme incubations were performed in accordance with the manufacturer's recommendations.
3
Quantifying Bnip1 Expression in Murine Tissues
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RNA from murine tissues (10 weeks old C57Bl/6 mice) or cultured skeletal cell types (at different stages of ex vivo differentiation) was isolated using the Macherey Nagel Nucleospin Kit (Macherey-Nagel).
Concentration and quality of RNA were determined using a NanoDrop ND-1000 system (NanoDrop Technology). For RT-PCR analysis 500 ng of RNA was reversed transcribed using Verso cDNA Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The quantitative expression analysis was performed using a StepOnePlus system and predesigned TaqMan gene expression assay (Bnip1: Mm01309803_m1, Thermo Fisher Scientific). Bnip1 expression levels were related to Gapdh expression for each tissue or cell culture sample set (n = 3).
Concentration and quality of RNA were determined using a NanoDrop ND-1000 system (NanoDrop Technology). For RT-PCR analysis 500 ng of RNA was reversed transcribed using Verso cDNA Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The quantitative expression analysis was performed using a StepOnePlus system and predesigned TaqMan gene expression assay (Bnip1: Mm01309803_m1, Thermo Fisher Scientific). Bnip1 expression levels were related to Gapdh expression for each tissue or cell culture sample set (n = 3).
4
Analyzing Hepatic Lipid Metabolism Genes
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Since biochemical results suggested that BDE-209 could be associated with disturbance of lipid metabolism through liver function, the total RNA was extracted from liver tissues of the control and cats treated with BDE-209 using TRI Reagent® (Sigma Life Science, USA) and cleaned up with NucleoSpin® kit (Macherey-Nagel, Germany), followed by cDNA synthesis using the ReverTra Ace® qPCR RT Master Mix with gDNA Remover (Toyobo Co., Ltd., Life Science Department, Osaka, Japan). S1 Table summarizes primer sets of genes involved in fat metabolism, including fatty acid elongase 6 (ELOVL6), stearoyl-CoA desaturase (SCD), acetyl-CoA carboxylase alpha (ACACA), and fatty acid synthase (FASN), as well as cytochrome P450 family 4 (CYP4). qRT-PCR (StepOnePlus Real-Time PCR system, Applied Biosystems, USA) was conducted using 10 μL of the PCR reaction mixture containing Fast SYBR Green Master Mix (Applied Biosystems, USA), forward and reverse primers (Thermo Fisher Scientific, Life Technologies Japan Ltd., Japan), and cDNA of each tissue. qPCR was conducted at 95°C for 20 s followed by 40 cycles of 95°C for 3 s and 60°C for 30 s. Quantification of the transcripts was performed by the ΔΔCT method [35 (link)] normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (ACTB) genes.
5
Distinguishing cDNA from Genomic DNA
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To differentiate between cDNA and genomic DNA, primers were designed at distinct sites of the exon–exon boundaries (Table S3 ). All primers were synthesized by Integrated DNA Technologies (Leuven, Belgium). Total cellular RNA was extracted using the RNA isolation NucleoSpin kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions. RT-PCR was performed using QuantiTect SYBR Green RT-PCR kit (Qiagen, Hilden, Germany), reaction capillaries (Roche Diagnostics, Mannheim, Germany) and a Light Cycler 1.5 (Roche Diagnostics). The amplification program started with the cDNA synthesis by a reverse transcription step at 50°C for 20 min, followed by pre-denaturation at 95°C for 15 min and 22–45 cycles of amplification (denaturation for 10 s at 95°C, annealing for 20 s at 59°C and elongation at 72°C for 40 s). For each reaction, the cycle threshold (Ct) was determined using the 2nd derivative method of the LightCycler 480 Software, release 1.5 (Roche). The relative gene expression levels were expressed as the difference in Ct values of the target gene and Gapdh. For knockout and wild type comparison analysis, knockout Ct values were calculated relative to Gapdh and wild-type cells according to the 2−ΔΔCt method (Schmittgen and Livak, 2008 (link)).
6
RNA Extraction and RT-PCR Analysis
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Total RNA was extracted from leaves (100 mg FW) of exposed (after 9 days of co-cultivation) and control plants. After 9 days of volatile treatment with 2 mM 6PP (see Effect of 6PP on Arabidopsis) or co-cultivation with T. asperellum IsmT5 (see Trichoderma—Plant Co-cultivation) seedlings were harvested, cells were homogenized by grinding with mortar and pestle in liquid nitrogen and RNA was enriched using the Nucleospin kit (Machery-Nagel, Düren, Germany). mRNA was reverse-transcribed into cDNA using the primers (Table S1 ) and SuperScript reverse transcriptase (Thermo Scientific Maxima Reverse Transcriptase) according to the protocol of the manufacturer. Gene-specific primers (Table S1 , obtained from Life Technologies, Carlsbad, USA) were used to amplify respective genes via PCR (Hippauf et al., 2010 (link)). The ubiquitin gene (AT4G05320) served as internal control.
7
Quantifying Mitochondrial DNA in Hepatocytes
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In order to quantify mitochondrial DNA (mtDNA) content of primary hepatocytes, we used NucleoSpin kit (Macherey‐Nagel) to extract total genomic DNA and then used real‐time PCR to detect two mtDNA‐specific sequences, namely cytochrome b (MT‐CYB) and 12S rRNA, using 18S as a nuclear DNA control. The primer sequences for PCR were listed in Table 1 .
8
Quantitative Expression Analysis of Dental MSCs
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Total RNA was extracted from 3 dental MSCs-loaded scaffolds at each time point with NucleoSpin kit (NucleoSpin RNA, Macherey-Nagel, Germany), as recommended by the manufacturer. Subsequently, cDNA synthesis was obtained with the iScriptTM cDNA Synthesis Kit (BioRad, United States) as recommended by the manufacturer. After cDNA synthesis reaction, quantitative real-Time PCR was carried out in mixture containing 1 μL of cDNA, 10 μM of each forward and reverse primers (Supplementary Table 2 ) and 10 μL of iTaqTM Universal SYBR® Green Supermix (BioRad, United States). qPCR experiments were run using an iQ5 (BioRad, United States) and analyzed with the iCycler IQ software (BioRad, United States). The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous assay control. Relative quantification of gene amplification by qPCR was performed using the cycle threshold (Ct) values and relative expression levels were calculated using the 2∧(−ΔΔCT) method. For each PCR, samples were analyzed in duplicate and three independent experiments were performed.
9
Plasmid Generation and Gene Deletion
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The primer sequences used in this study for generating plasmids or confirming the deletion of target genes are described in Additional file 4 : Table S1. The primers were designed by In Silico Molecular Cloning (IMC, In Silico Biology, Inc., Yokohama, Japan), plasmid Editor Software or Oligo Primer Analysis Software ver.7 (Molecular Biology Insights, Colorado Springs, CO, USA). GoTaq DNA polymerase (Promega, Fitchburg, WI, USA) was used for colony-direct PCR of E. coli transformants, and KOD-plus-Neo DNA polymerase (Toyobo Co. Ltd., Osaka, Japan) was used for cloning of B. longum genomic DNA, as described by the manufacturer’s protocol. The PCR products were analyzed by 1% (w/v) agarose gel electrophoresis with TAE buffer. For cloning, the target fragments of the PCR products were extracted from the gel using a NucleoSpin kit (MACHEREY–NAGEL, Düren, Germany). The PCR conditions using KOD-plus-Neo DNA polymerase were as follows: 94 °C for 2 min, 25 cycles of 98 °C for 10 s, 57 °C or 60 °C for 30 s, and 68 °C for 1 min. The PCR conditions using GoTaq DNA polymerase were as follows: 95 °C for 2 min, 30 cycles of (95 °C for 1 min, 57 °C or 60 °C for 1 min, and 72 °C for 1 min), then 72 °C for 10 min.
10
Fungal DNA Extraction from Lisianthus
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Genomic DNA from F. oxysporum isolates from lisianthus was extracted from approximately 100 mg of 7 days grown mycelium scraped from petri dishes, by using Tissue Lyzer (Qiagen, Hilden, Germany) and the NucleoSpin kit (Macherey Nagel, Duren, Germany), according to the manufacturer's protocol, adding 10 μl Proteinase K solution (10 mg/ml) and 10 μl RNAse A (12 mg ml -1 ) to the lysis buffer in each tube. For every isolate, DNA was extracted in duplicate. DNA extracted were stored at -20°C.
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