The V9 region of the 18S rRNA gene was amplified using universal primers (1380F: 5′-CCCTGCCHTTTGTACACAC; 1510R: 5′-CCTTCYGCAGGTTCACCTAC) [21 (link)]. We used 200 ng DNA of each sample for the polymerase chain reaction (PCR). The amplicons were verified by 2% agarose gel electrophoresis and then mixed into equal amounts based on product concentrations. The Gel Extraction Kit (QIAGEN, Germany) was used to purify the amplicons, then the amplicons were pooled to construct the libraries using the TruSeq® DNA PCR-free Sample Preparation Kit. The barcode was ligated to the 5′ ends of primers to distinguish each sample. After Qubit and qPCR quantitative detection, paired-end sequencing of the amplicons was performed on a HiSeq2500 PE250 sequencer platform (Novogene, Beijing, China).
Truseq dna pcr free sample preparation kit
Manufactured by Qiagen
Sourced in Germany
The TruSeq® DNA PCR-Free Sample Preparation Kit is a laboratory equipment product designed for the preparation of DNA samples. It enables DNA sample preparation without the need for PCR amplification. The kit provides a streamlined workflow for DNA library construction.
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Lab products found in correlation
Gel extraction kit, by Qiagen (4 mentions)
Gel recovery kit, by Qiagen (4 mentions)
Novaseq 6000 platform, by Illumina (2 mentions)
Phusion high fidelity pcr master mix with gc buffer, by New England Biolabs (2 mentions)
Gc buffer, by New England Biolabs (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Qubit 2, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Novaseq platform, by Illumina (1 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Qubit 2.0 fluorometer, by Agilent Technologies (1 mentions)
9 protocols using truseq dna pcr free sample preparation kit
1
18S rRNA gene amplification and sequencing
2
16S rRNA V3 Region Library Preparation
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The library targeting the V3 region of 16S rRNA was constructed by using DNA samples separated from the cecum contents. In brief, individual DNA samples were amplified by PCR using a specific primer with Barcode and Phusion High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs, Beverly, MA). Then PCR amplicon was detected by electrophoresis using 2% agarose gel. The gel extraction kit (Qiagen, GmbH, Germany) was used to isolate the purpose gene fragments. And then, both library concentration and an exact product size were measured using TruSeq DNA PCR-Free Sample Preparation Kit through a quantitative PCR and Qubit. The PCR-free library was constructed based on the illumina Nova sequencing platform. NovaSeq6000 was used for on-board sequencing following qualified the library, and 140 bp paired-end reads were produced, and the raw-sequence data of the whole experiment have been submitted to https://submit.ncbi.nlm.nih.gov/subs/bioproject/SUB8069709/overview .
3
Amplicon Sequencing of Bacterial 16S rDNA
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Diluted genomic DNA was used as the template, and the bacterial V4 hypervariable region of 16S rDNA was amplified by PCR using specific primers with barcodes, Phusion® High-Fidelity PCR Master Mix with GC Buffer from New England Biolabs, and a high-efficiency high-fidelity enzyme according to the selection of the sequencing region. The primer pair was 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The PCR products were multiplexed in a single pool in equimolar amounts and then detected by electrophoresis using 2% agarose gel electrophoresis after full mixing. The target bands were recovered using a gel recovery kit provided by Qiagen, and a TruSeq® DNA PCR-Free Sample Preparation Kit was used for amplicon library preparation. All PCR reactions were carried out in 30 μL reactions, 0.2 μM of forward and reverse primers, and about 10 ng template DNA. Thermal cycling consisted of initial denaturation at 98°C for 1 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and elongation at 72°C for 30 s. Finally 72°C for 5 min. After quantification of the library with a Qubit 2.0 Fluorometer (Thermo Fisher Scientific Inc.) and quantitative PCR, sequencing was conducted using a NovaSeq6000 platform (Illumina, San Diego, CA, United States).
4
Bacterial DNA Extraction and 16S rRNA Amplicon Sequencing
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After thawing the frozen samples at room temperature, bacterial genomic DNA was extracted by the cetyltrimethylammonium bromide (CTAB) method [14 (link)]. Agarose gel electrophoresis was used to assess the purity and concentration of DNA. Then suitable amounts of sample DNA were taken and diluted to 1 ng/µL by using sterile water. Subsequently, 1% agarose gel electrophoresis was used to detect the integrity of the DNA, and both the purity and concentration of each DNA sample were measured by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The isolated DNA was frozen at −20 °C until the next experiment. The V4 region of the 16S rRNA was amplified by using the primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The target bands were recovered using a gel recovery kit provided by Qiagen, and a TruSeq® DNA PCR-Free Sample Preparation Kit was used for amplicon library preparation. PCR reagents and conditions were the same as those described by Winther et al. [16 (link)]. The final library concentration was quantified using Qubit 2.0 (ThermoFisher Scientific, Waltham, MA, USA) and quantitative PCR, and the sequencing was on a NovaSeq6000 platform (Illumina, San Diego, CA, USA).
5
Amplification and Sequencing of Marker Genes
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To prepare samples for sequencing, total genomic DNA was extracted using the CTAB method and checked for concentration and purity with 1% agarose gel. DNA was diluted to 1 ng/μL using sterile water. Next, specific regions of the 16S rRNA/18SrRNA/ITS genes (such as 16S V4/16S V3/16S V3-V4/16S V4-V5, 18S V4/18S V9, ITS1/ITS2, Arc V4) were amplified using specific primers with a barcode.
PCR reactions utilized Phusion® High-Fidelity PCR Master Mix and 10 ng of template DNA, followed by thermal cycling and 2% agarose gel detection. PCR products were purified using a Qiagen Gel Extraction Kit, and sequencing libraries were generated with the TruSeq® DNA PCR-Free Sample Preparation Kit, adding index codes. Library quality was assessed using a Qubit@ 2.0 Fluorometer and an Agilent Bioanalyzer 2,100 system. Sequencing was performed on an Illumina NovaSeq platform, producing 250 bp paired-end reads.
PCR reactions utilized Phusion® High-Fidelity PCR Master Mix and 10 ng of template DNA, followed by thermal cycling and 2% agarose gel detection. PCR products were purified using a Qiagen Gel Extraction Kit, and sequencing libraries were generated with the TruSeq® DNA PCR-Free Sample Preparation Kit, adding index codes. Library quality was assessed using a Qubit@ 2.0 Fluorometer and an Agilent Bioanalyzer 2,100 system. Sequencing was performed on an Illumina NovaSeq platform, producing 250 bp paired-end reads.
6
Amplification and Sequencing of 16S rDNA
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The genomic DNA was extracted from intestinal contents by cetyl trimethyl ammonium bromide (CTAB) or sodium dodecyl sulfonate (SDS) methods. The purity and concentration of the DNA samples were examined by agarose gel electrophoresis. The purified DNA samples were individually diluted to 1 ng/μL with water. Using the diluted genomic DNA as a template, PCR was performed with the primers 341F: 5′-CCTAYGGGRBGCASCAG-3′ and 806R: 5′-GGACTACNNGGGTATCTAAT-3′ to amplify a specific region of the 16S rDNA gene. The PCR system contained specific primers with barcodes, Phusion® High-Fidelity PCR Master Mixture with GC Buffer, and high-efficiency high-fidelity polymerase from New England Biolabs. PCR products were checked on 2% agarose gel by electrophoresis. According to the concentrations of PCR products, PCR products of the same amount for all the samples were mixed. The target bands were recovered for library construction using a gel recovery kit (Truseq ® DNA PCR-Free Sample Preparation Kit) provided by Qiagen. The constructed library was quantified by Qubit. When the qualified library was constructed, NovaseQ 6000 was then used for sequencing analysis.
7
Rumen microbiome profiling by 16S rRNA sequencing
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Rumen content samples were thawed and microorganism DNA extracted by the CTAB method. Concentration and puri cation of DNA were carried out by 1% agarose gel electrophoresis for 40 min (voltage 100 V).
V3 and V4 regions of bacterial 16S rRNA genes were ampli ed using the primer sequence(5'-3')341F:CCTAYGGGRBGCASCAG.806R: GGACTACNNGGGTATCTAAT. High-e ciency, highdelity PCR enzymes and Phusion® High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs) were used to maximize PCR ampli cation e ciency and accuracy. Equal amounts of PCR reagents and samples were thoroughly mixed and PCR products detected by 2% agarose gel electrophoresis. Target strips were recovered using a Qiagen gel recovery kit and a TruSeq® DNA PCR-Free Sample Preparation Kit was used to construct the library. The library was quanti ed using Qubit and Q-PCR and sent to Beijing NovaSeq PE250 for 16S rRNA high-throughput sequencing.
V3 and V4 regions of bacterial 16S rRNA genes were ampli ed using the primer sequence(5'-3')341F:CCTAYGGGRBGCASCAG.806R: GGACTACNNGGGTATCTAAT. High-e ciency, highdelity PCR enzymes and Phusion® High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs) were used to maximize PCR ampli cation e ciency and accuracy. Equal amounts of PCR reagents and samples were thoroughly mixed and PCR products detected by 2% agarose gel electrophoresis. Target strips were recovered using a Qiagen gel recovery kit and a TruSeq® DNA PCR-Free Sample Preparation Kit was used to construct the library. The library was quanti ed using Qubit and Q-PCR and sent to Beijing NovaSeq PE250 for 16S rRNA high-throughput sequencing.
8
PCR Product Purification and Sequencing
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The PCR products were recovered using the Qiagen gel extraction kit (Qiagen), and the TruSeq DNA PCR-Free Sample Preparation kit was used to construct the library. The constructed library was quantified by Qubit and quantitative (q) PCR. After the library was approved, NovaSeq6000 was used for computer sequencing.
9
Gel Extraction and Library Preparation
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The PCR product was detected by electrophoresis with 2% agarose gel. According to the concentration of PCR product, the samples were mixed equally, and then the PCR products were detected by agarose gel electrophoresis with 2% agarose gel. The gel recovery kit provided by Qiagen company was used to recover the target band.
(3) Library construction and sequencing Library construction used TruSeq® DNA PCR-Free Sample Preparation Kit (Building Database Kit). The library was quanti ed by qubit and Q-PCR, and after the library was quali ed, it was sequenced by novaseq6000.
(3) Library construction and sequencing Library construction used TruSeq® DNA PCR-Free Sample Preparation Kit (Building Database Kit). The library was quanti ed by qubit and Q-PCR, and after the library was quali ed, it was sequenced by novaseq6000.
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