The collected material was ground in a mortar chilled to −70 °C. Then DNA was isolated using the DNeasy PowerSoil Kit (QIAGEN, Hilden, Germany). The DNA was then purified using the Anti-Inhibitor Kit (A&A Biotechnology, Gdynia, Poland). Total DNA was sent for sequencing and preparation of OTU (operational taxonomic unit) libraries at Genomed SA (Warsaw, Poland). Fungal community analysis was performed based on the Internal transcribed spacer 1 (ITS1) region using specific primers ITS1FI2 5′-GAA CCW GCG GAR GGA TCA-3′ [62 (link),63 (link)] and 5.8S 5′-CGC TGC GTT CTT CAT CG-3′ [64 (link)]. PCR was performed using Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA, USA). Reaction conditions were as recommended by the manufacturer. Sequencing was performed on a MiSeq sequencer in paired-end (PE) technology. Negative samples (without DNA) were also sequenced to remove artifacts.
Anti inhibitor kit
Manufactured by A&A Biotechnology
Sourced in Poland
The Anti-Inhibitor Kit is a laboratory equipment designed to detect and quantify the presence of inhibitors in various samples. It provides a reliable and efficient means for researchers and scientists to analyze the levels of inhibitors, which can be crucial in various applications such as enzyme activity studies, protein purification, and diagnostic assays.
Automatically generated - may contain errors
Lab products found in correlation
Dneasy powersoil kit, by Qiagen (1 mentions)
Q5 hot start high fidelity 2x master mix, by New England Biolabs (1 mentions)
Genomic mini ax stool kit, by A&A Biotechnology (1 mentions)
Genomic mini ax bacteria kit, by A&A Biotechnology (1 mentions)
Fastprep 24 apparatus, by MP Biomedicals (1 mentions)
4 protocols using anti inhibitor kit
1
Fungal Community Profiling via ITS1 Sequencing
2
Identifying 1BL/1RS Translocation in Wheat Hybrids
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The leaves of three-week-old seedlings of spelt, common wheat and wheat hybrids were collected. DNA was isolated with a ready-to-use Genomic Micro AX Plant Gravity Kit (A&A Biotechnology, Poland). The quantity and quality of DNA was determined with a spectrophotometer (nanoMaestro Gen, Poland) at 260 nm and 280 nm wavelength. Extracted DNA was additionally purified before further analysis with the use of the Anti-Inhibitor Kit (A&A Biotechnology, Poland). The 1BL/1RS translocation in investigated parental components and common wheat-spelt hybrids was identified by PCR according to the method described by Iqbal and Rayburn [20 (link)] with specific primers JO71F1 5’-TAAGCCGTAAAGCATGGTGCAC-3’ and J07IR1 5’-CTTCAACGAAAT GTT TTC CTC TTC-3’. Total reaction volume was 20 μl.
3
Optimized DNA Extraction from Fecal Samples
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Before DNA extraction, the material was subjected to three cycles of freezing at –70 °C and thawing at 30 °C in a water bath to destroy the cyst wall and improve the efficiency of DNA extraction. Genomic DNA was then extracted from approximately 100 mg of a fecal sample using the Genomic Mini AX Stool kit (A&A Biotechnology, Gdansk, Poland) according to the original protocol. All of the PCR templates were also treated with the Anti-Inhibitor Kit (A&A Biotechnology, Gdansk, Poland), which removes polyphenolic PCR inhibitors using specific absorption particles, thereby removing factors that could interfere with the PCR. The extracted DNA was stored at −20 °C for further analysis.
4
Efficient Bacterial DNA Extraction and Sequencing
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The application of mutanolysin and lysozyme in the Genomic Mini AX Bacteria +” kit (A&A Biotechnology, s. c., Gdańsk, Poland) ensured the effective extraction and precipitation of the genomic DNA, resulting from the digestion of cell walls of the bacteria particularly resistant to the lysis that preceded the determination of DNA. The mechanical lysis was conducted using a FastPrep-24 apparatus (MP Biomedicals LLC, Solon, OH, USA). The next stage included an additional purification using an Anti-Inhibitor Kit (A&A Biotechnology s. c., Gdańsk, Poland). Bacterial DNA was determined in the samples by the colorimetric method and confirmed using Real-Time PCR (A&A Biotechnology s. c., Gdańsk, Poland). Based on universal starters of the PCR mixture containing 1055F in the presence of SYBR pigment, the sequencing of the gene encoding the amplicon 16S sequences was conducted, based on the V3-V4 hypervariable region. The bio-informatic analysis was conducted by Genomed SA (Warsaw, Poland) using a MiSeq v2 Illumina sequencer (Illumina, Inc., San Diego, CA, USA) by assigning operational taxonomic units (OUT) to the sequencing reads on the 16S RNA gene amplicon sequencing in accordance with the taxonomic affiliation to the genus level.
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