The V3-V4 hypervariable region of 16S rRNA genes were amplified using universal primers with overhang adapters (338 Fin TTCCCTACACGACGCTCTTCCGATCT-ACTCCTACGGRAGGCAGCAG; 806 Rin GAGTTCCTTGGCACCCGAGAATTCCA-GGACTACHVGGGTWTCTAAT) by the Phusion High Fidelity PCR Master Mix with HF Buffer (New England Biolabs, Hitchin Herts, UK). The PCR reactions were performed as follows: initial denaturation at 94 °C × 2 min; 25 cycles of denaturation at 94 °C × 30 s, annealing at 56 °C × 30 s, elongation at 72 °C × 45 s; and final extension at 72 °C × 2 min and held at 10 °C × 10 min. After purification using the AXYGEN AxyPrep DNA Gel Extraction Kit (Axygen Scientific, Union City, CA, USA), the libraries were then normalized according to Qubit3.0. The barcoded 16S rRNA gene was sequenced on the Illumina MiSeq sequencing platform (Illumnia, Inc., San Diego, CA, USA) at TinyGene Bio-Tech (Shanghai), Co., Ltd. (Shanghai, China), using a 2 × 300 cycle V3 kit, following standard Illumina sequencing protocols.
Axygen axyprep dna gel extraction kit
Manufactured by Corning
Sourced in United States
The AXYGEN AxyPrep DNA Gel Extraction Kit is a laboratory product designed to efficiently extract and purify DNA fragments from agarose gels. It utilizes a silica-based membrane technology to bind and recover DNA, allowing for the removal of contaminants and salts. The kit provides a simple and reliable method for extracting DNA from gel slices, making it a useful tool for various molecular biology applications.
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Lab products found in correlation
Miseq sequencing platform, by Illumina (2 mentions)
Phusion high fidelity pcr master mix with hf buffer, by New England Biolabs (2 mentions)
Miseq instrument, by Illumina (2 mentions)
Quantifluor st blue fluorescence quantification system, by Promega (1 mentions)
Miseq system, by Illumina (1 mentions)
Reaction buffer, by Takara Bio (1 mentions)
Q5 high fidelity dna polymerase, by Takara Bio (1 mentions)
Miseq pe250 sequencer, by Illumina (1 mentions)
Nanodrop nd 1000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Hf buffer, by New England Biolabs (1 mentions)
Phusion high fidelity pcr master mix, by New England Biolabs (1 mentions)
One step primescript rt pcr kit, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Pmd19 t vector, by Takara Bio (1 mentions)
Taq version 2.0 plus dye, by Takara Bio (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Linear accelerator, by Rad Source (1 mentions)
9 protocols using axygen axyprep dna gel extraction kit
1
16S rRNA Gene Amplification and Sequencing
2
Amplification and Sequencing of Microbial 16S rDNA
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The metagenomic DNA was diluted and used as a template for the V3 + V4 variable region of bacterial 16S rDNA. PCR products were detected by 2% agarose gel electrophoresis and purified by the Axygen®AxyPrep DNA gel extraction kit (Axygen Scientific Inc., Union City, CA, USA). And the quantified by QuantiFluorTM-ST Blue Fluorescence Quantification System (Promega), in which the corresponding proportion was mixed according to the sequencing volume requirement of each sample. A TransStart Fastpfu, DNA Polymerase 20-μL reaction system, was used, including 2.0 μL of 10 × buffer, 2.0 μL of 2.5 m MdNTPs, 0.8 μL of forwarding primer (5 μmol/L), 0.8 μL of reverse primer (5 μmol/L), 0.2 μL of Taq polymerase, 0.2 μL of BSA, 10 μL of template DNA, and 20 μL of ddH2O (23 (link)). The PCR products were then sequenced by the Illumina MiSeq sequencing platform (Illumina, San Diego, CA, USA) by Wuhan Fraser Genetic Information Co., Ltd.
3
Amplification and Sequencing of Gut Microbiome
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The DNA isolated from fecal samples was used as the template for the amplification of the 16S rRNA V3-V4 region. The universal primers used were F (5’-NNNNNNN ACTCCTACGGGAGGCAGCA-3’) and R (5’-NNNNNNN GGACTACVSGGGTATCTAAT-3’), with the NNNNNNN being unique seven-base barcode used to tag each PCR product. The PCR reaction was performed according to the touchdown protocol[18 (link)] in a system of 25 μL containing 5.0 μL 5 × reaction buffer (TaKaRa, Dalian, China), 5.0 μL 5 × high GC buffer (TaKaRa, Dalian, China), 0.5 μL dNTPs (10 mmol/L) mixture , 1.0 µL forward primer (10 µmol/L), 1.0 μL reverse primer (10 µmol/L), 0.25 μL Q5 high-fidelity DNA polymerase (5 U/uL, TaKaRa, Dalian, China), and 1 μL DNA template. Each PCR product was purified by 2% agarose gel electrophoresis. DNA was isolated using the Axygen Axy Prep DNA Gel Extraction kit (Axygen, Shanghai, China). The sequencing was finished with the help of the Illumina Miseq System (Illumina).
4
Amplification and Sequencing of Gut Microbiome
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Fecal samples were stored at −80°C until DNA extraction. Total genomic DNA was extracted from each sample using a QIAamp DNA Stool Mini Kit in accordance with the manufacturer's instructions. PCR amplification and MiSeq sequencing were performed as previously described [20 (link)]. In brief, the V4–V5 regions of bacterial 16S rDNA were amplified using the Phusion High-Fidelity PCR Master Mix with HF buffer (New England Biolabs, UK). Barcode-indexed PCR primers 515F and 926R were used. Amplicon libraries were purified using the AXYGEN AxyPrep DNA Gel Extraction Kit (Axygen Scientific, Union City, CA, USA), normalized via FTC-3000TM real-time PCR, and sequenced using a MiSeq instrument (Illumina) using a 2 × 300-cycle V3 kit.
5
Soil Microbiome Analysis using 16S rRNA
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The EZNATM Soil DNA Kit (Omega, United States) was used to extract the DNA of the soil at a UV-sterilized ultraclean bench. We used 1% (m/v) agarose gel electrophoresis to analyze the eluted DNA samples and used a NanoDrop® ND-1000 UV–Vis spectrophotometer (Thermo Fisher Scientific, United States) to measure the DNA concentration. The V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified using Liu et al. (2021) (link) method with the primer (338F 5′- ACTCCTACGGGAGGCAGCAG-3′ and 806R 5′-GGACTACHVGGGTWTCTAAT-3′). Amplification was performed with predenaturation for 3 min at 95°C, followed by 26 cycles of 30 s at 95°C, 30 s at 55°C, and 45 s at 72°C, and final extension for 10 min at 72°C. PCR amplicons were detected by electrophoresis on 2% (w/v) gels, and the Axygen® AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, United States) was used to recover the target fragments. The purified amplicons were sequenced on the Illumina MiSeq PE250 sequencer.
6
Bacterial 16S rDNA Amplification and Sequencing
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Samples were stored at −80°C until the DNA is extracted. QIAamp DNA Stool Mini Ki was used to extract all genomic DNA from each sample. As mentioned earlier, PCR amplification and MiSeq sequencing were performed. In summary, Phusion High-Fidelity PCR Master Mix and HF buffer (New England Biolabs, UK) were used to amplify the V4-V5 region of the bacterial 16S rDNA. Barcode index PCR primers 515F and 926R were used. The amplicon library with the AXYGEN AxyPrep DNA Gel Extraction Kit (AXYGEN Scientific, Union City, CA, USA) was purified, normalized with FTC-3000TM Real-Time PCR, using the MiSeq instrument (Illumina), using 2 × 300 cycles of V3 reagents. The cassette is sequenced.
7
Extraction and Sequencing of H9N2 HA Gene
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Total RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions, then amplified by one-step RT-PCR with a PrimeScript One-Step RT-PCR Kit (Takara Bio, China) as previously described [4 (link)]. The specific primer set designed for amplification of the H9N2 HA gene was HA-F: 5'-CAAGATGGAAGTAGTATCACT-3' and HA-R: 5'-TTGCCAATTATATACAAATGT-3'. RT-PCR was conducted by subjecting the samples to 50℃ for 30 min, 94℃ for 2 min and then 30 cycles of 94℃ for 40 sec, 53℃ for 40 sec and 72℃ for 2 min, followed by final extension at 72℃ for 10 min. PCR products were purified using an Axygen AxyPrep DNA Gel Extraction Kit (Corning, USA) according to the manufacturer's instructions, after which the product was cloned into pMD-19T vector (Takara Bio) for sequencing. The purified recombinant plasmids were sequenced by Invitrogen Trading (Shanghai).
8
Validation of Chimeric RNA Candidates
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Candidate chimeric RNAs were validated by RT-PCR. Specific primer pairs for the candidates were designed, with each primer flanking the junction site. All primers used for detection are listed in Supplementary Table S1 . RT-PCR experiments were performed with TaKaRa Taq™ Version 2.0 plus dye (Shiga, Japan). Following RT-PCR and gel electrophoresis, Axygen® AxyPrep DNA Gel Extraction Kit (Corning, NY, USA) was used for DNA purification and followed by Sanger sequencing at Sangon Biotech.
9
Characterization of HaCaT Keratinocyte Cell Line
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Human keratinocyte HaCaT (cat. no. iCell-h006, iCell Bioscience Inc.) and human skin fibroblast WS1 (cat. no. CRL-1502T; ATCC) cells were maintained in DMEM supplemented with 10% FBS and 100 U/ml penicillin-streptomycin at 37˚C and a 5% CO2 atmosphere. For transfection, cells were transfected by Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) with viral particles. Cells were exposed to a single dose (20 Gy) of X-rays using the linear accelerator (RadSource) at a dose rate of 1.15 Gy/min. The HaCaT cell line was obtained from iCell Bioscience Inc. DNA was extracted with Axygen genome extraction kit (cat. no. AP-GX-250; Axygen® AxyPrep DNA Gel Extraction Kit; Corning, Inc.) and amplified with 21-str amplification scheme. STR loci and sex gene Amelogenin were detected on an ABI 3730XL genetic analyzer (Applied Biosytems; Thermo Fisher Scientific, Inc.). The results showed that the cell line was completely matched by DNA typing in cell line retrieval and the cell name was HACAT and the cell number was 771 according to DSMZ database. No multiple alleles were found in this cell line.
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