Soil DNA was extracted according to Mandakovic et al. (2018a (link)) with some modifications. Total DNA from soils was extracted from 10 g of each sample using the NucleoSpin® Food kit (Macherey-Nagel), following the manufacturer's instructions, and the CTAB extraction buffer published by Zhou et al. (1996 (link)). Microbial 16S rRNA genes were amplified according to Mandakovic et al. (2018b (link)) without modifications. DNA libraries were constructed following the TruSeq DNA sample preparation (Illumina, USA) protocol. Sequencing was performed by MrDNA Next Generation Sequencing Service Provider (Shallowater, Texas, USA) on an Illumina MiSeq platform in an overlapping 2 × 300 bp configuration, to obtain a minimum throughput of 40,000 sequences (reads) per sample.
Nucleospin food kit
Manufactured by Macherey-Nagel
Sourced in Germany
The NucleoSpin Food kit is a DNA/RNA extraction and purification kit designed for the isolation of nucleic acids from various food samples. It provides a reliable and efficient method for extracting and purifying DNA or RNA from a wide range of food matrices.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 4.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Synergy ht, by Agilent Technologies (2 mentions)
Gen5 data analysis software version 2, by Agilent Technologies (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Miseq platform, by Illumina (1 mentions)
Truseq dna sample preparation, by Illumina (1 mentions)
Quantus fluorometer, by Promega (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Versafluor fluorometer, by Bio-Rad (1 mentions)
Premix taq dna polymerase, by Takara Bio (1 mentions)
Lightcycle 480, by Roche (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Gel doc ez imager, by Bio-Rad (1 mentions)
Gelred, by Biotium (1 mentions)
Image lab software version 5, by Bio-Rad (1 mentions)
Gel doc ez system, by Bio-Rad (1 mentions)
Take3 micro volume plate accessory, by Agilent Technologies (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Smrtbell template prep kit 1, by Pacific Biosciences (1 mentions)
Qubit system, by Thermo Fisher Scientific (1 mentions)
35 protocols using nucleospin food kit
1
Soil Microbial DNA Extraction and Sequencing
2
Salmonidae Genetic Reference Database
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Authenticated Salmonidae tissues from nine species involved in the creation of the SEAFOOD TOMORROW genetic reference database (Deconinck et al., 2020; (link)SeafoodTomorrow, n.d.) were used to develop a specific ddPCR assay for Salmo salar (Table 1). These tissues were stored on ethanol at -20 °C until extraction. Total DNA was isolated from 200 mg of muscle tissue (or food product) using the NucleoSpin® Food kit (Macherey -Nagel GmbH & Co. KG, Düren, Germany) following the manufacturer's instructions. Total DNA was quantified using a Quantus TM fluorometer (Promega, Madison, USA), following the manufacturer's protocol. DNA extractions were stored at -20 °C until further use.
Table 1 Salmonidae used to test the specificity of the ddPCR assay, including the scientific and common name, the number of the specimen and the sample origin. Fishtrace ("FishTrace," n.d.) and GenBank (NCBI, Bethesda, USA) (Supplementary Table S1).
Sequences were aligned and trimmed appropriately using Mega X version 10.0.5 (Tamura et al., 2007) .
Table 1 Salmonidae used to test the specificity of the ddPCR assay, including the scientific and common name, the number of the specimen and the sample origin. Fishtrace ("FishTrace," n.d.) and GenBank (NCBI, Bethesda, USA) (Supplementary Table S1).
Sequences were aligned and trimmed appropriately using Mega X version 10.0.5 (Tamura et al., 2007) .
3
Comprehensive DNA Extraction and Evaluation
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The genomic DNA used in this study was extracted with a CTAB DNA extraction method modified from ISO 21571 (International Organization for Standardization, 2005 ) (for soybean, oilseed rape and with an additional phenol-chloroform purification step for cotton when necessary) or a NucleoSpin® Food kit (Macherey-Nagel, for maize). In cases of suboptimal recovery or inhibition, the NucleoSpin® Food kit (for oilseed rape) or the foodproof® Sample Preparation kit III (Biotecon Diagnostics, for cotton) were used. The integrity of the extracted genomic DNA was tested by electrophoresis on a 1% [w/v] agarose gel stained with ethidium bromide, while the absence of PCR inhibitors was assessed through a real-time PCR inhibition run as described by Zel et al. (Žel et al., 2008 ). Only pure, non-inhibited, high molecular weight DNA was used. The extractions were done at least in duplicate with CRMs containing two different GM events per each crop; DNA samples were maintained at 4 °C for the duration of the experiments, then stored at −20 °C.
4
Diverse DNA Extraction Protocols
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DNA was extracted from 200 mg of starting material; different methods were used based on the matrix from which the DNA was to be extracted: CTAB DNA extraction method was used for soybean (modified from ISO 21571 [35 ]), NucleoSpin Food kit (Macherey-Nagel GmbH, üren, Germany) for maize, and Biotecon foodproof Sample Preparation Kit III (Biotecon Diagnostics GmbH, Potsdam, Germany) for the muesli mix and strawberries. Rice DNA was extracted from leaves as described elsewhere [34 (link)]. The integrity of the extracted genomic DNA was controlled by agarose gel electrophoresis, followed by DNA quantification using Quant-iT PicoGreen dsDNA Assay kit (Life Technologies, Molecular Probes, Eugene, Oregon, USA) and a Bio-Rad VersaFluor fluorometer.
5
CTAB and Nucleospin DNA Extraction
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Genomic DNA from all plant materials and the real-life samples was extracted using the CTAB method (ISO21571 [16 ] with minor modifications), except for Bt176 and Bt11, where the Nucleospin Food Kit (Macherey-Nagel, Düren, Germany) was used. The sample intake was 200 mg or 1 g per isolation for the CTAB extractions and 200 mg per isolation for the Nucleospin Food Kit. Quantity of DNA used in the reactions described below was from 100 to 10 ng per reaction.
6
Exine Rupture Treatment Optimization
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Fourteen of the above species (Cupressus sempervirens L. was eliminated due to presence of non-pollen plant material in sample) were suspended as single species in sterile 50% glycerol and 10 replicate counts of 10 μL were performed in order to create 18 replicate aliquots of 10,000 pollen grains for each species. From these 18 replicates, 6 treatments with 3 replicates (Table 3 ) were created by implementing the optimal bead mill conditions determined above. DNA extraction was performed with Macherey Nagel Nucleospin food kit following the instructions for honey with 400 μL lysis buffer and a final elution volume of 50 μL. Mixed species DNA extractions were created by combining equal volumes of each single species extractions for a final creation of 3 replicates of each of the 6 exine rupture treatments. The DNA quantity of these mixed species extractions were quantified with Qubit™ 4 fluorometer using the dsDNA HS Assay kit.
7
Bifidobacterium longum subsp. longum Strain Identification
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In this study, 22 samples of Bifidobacterium longum subsp. longum strain UABl-14™ were used. Four of these samples were mono-strain samples and 18 were multi-strain samples acquired directly from manufacturers (Table 1 ). Additionally, reference samples from 30 probiotic strains were included in this study as non-targets (Table 1 ). The samples were collected from various probiotic manufacturers in USA and Canada. DNA extraction was performed using NucleoSpin Food kit (740945.50, Macherey Nagel, Germany), followed by DNA quantification using Qubit 4.0 FLuorometer (Q33238, Life technologies).
8
DNA Extraction from Enriched Broth
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After 24 h of enrichment, 1 ml of the enriched broth was transferred into a 1.5-ml micro-centrifuge tube, centrifuged for 10 min at 6000×g at room temperature, and the supernatant was discarded. DNA was extracted from the pellet with the Nucleospin Food Kit (Macherey-Nagel®, Düren, Germany) according to the manufacturer’s recommendations.
9
Maize Kernel DNA Extraction and Quantification
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From each of the samples, the cobs were manually shelled, the kernels mixed and grounded using a GRINDOMIX GM 200 blade mill (Retsch GmbH, Haan, Germany). 200-250 mg of the resulting powder was used for the DNA extraction with the NucleoSpin ® Food kit (Macherey-Nagel GmBH, Düren, Germany), conducting one measurement per sample, as described in Corujo (2016) .
DNA quantification was performed using a spectrophotometer (NanoDrop, Wilmington, USA), reading the D.O. at 260 nm. The qPCRs were carried out using a Light Cycle 480 real-time PCR Instrument (Roche Holding AG, Basel, Switzerland), fluorescence monitored with Light Cycle 480 software v 1.5.0 (Roche Holding AG, Basel, Switzerland). Detection and quantification of the MON810 event was conducted by the validated JCR method by Shindo et al. (2002) (link), starting from 100 µg DNA, and using Premix Taq™ DNA Polymerase (TaKaRa Taq™ Version 2.0) (Takara Bio Inc., Shiga, Japan).
DNA quantification was performed using a spectrophotometer (NanoDrop, Wilmington, USA), reading the D.O. at 260 nm. The qPCRs were carried out using a Light Cycle 480 real-time PCR Instrument (Roche Holding AG, Basel, Switzerland), fluorescence monitored with Light Cycle 480 software v 1.5.0 (Roche Holding AG, Basel, Switzerland). Detection and quantification of the MON810 event was conducted by the validated JCR method by Shindo et al. (2002) (link), starting from 100 µg DNA, and using Premix Taq™ DNA Polymerase (TaKaRa Taq™ Version 2.0) (Takara Bio Inc., Shiga, Japan).
10
Comprehensive Probiotic Strain Database
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A total of 182 probiotic products, containing a total of 520 strains in 470 species, were collected from various probiotic manufacturers in USA and Canada (Supplementary Table S1 ). Among them, 45 products were finished products in their final delivery form and 137 products were powder materials ready for formulation into their final delivery form. Samples were stored either frozen or at room temperature, following the recommended storage conditions. DNA was extracted from all probiotic samples using NucleoSpin Food kit (740945.50, Macherey Nagel, Germany), according to manufacturer’s instructions. DNA was quantified using Qubit 3.0 Fluorometer and was stored in a − 20°C freezer until use.
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