Universal primers 515 F and 806R were used for PCR amplification of the V4 hypervariable region of the 16S rRNA gene according to the procedure of Caporaso et al. [35 (link)]. The 806R primer included a unique 12 bp sequence tag to barcode each sample. The V4 region of the 16S rRNA genes was amplified in 96-well microtitre plates using Phusion High-Fidelity DNA polymerase (Thermo Fisher, USA) and 50 ng template DNA in a total reaction volume of 50 μl. Reactions were run in a DNA Engine Tetrad 2 thermo cycler (Bio-Rad, USA) using the following cycling parameters: 30 s at 98 °C, followed by 25 cycles of 10 s at 98 °C, 15 s at 55 °C, and 15 s at 72 °C, with a final step of 10 min at 72 °C. Negative controls without template were included for each barcoded primer pair. Amplicons were confirmed by gel electrophoresis (2% E-gel) and quantified using the Quant-iT™ PicoGreen® dsDNA assay (Life Technology, USA). Equimolar amounts (100 ng) of each of 278 PCR amplicons were mixed in a single tube. Primers and reaction buffer were removed from the pool of samples using the AMPure kit (Agencourt, USA). The purified amplicon mixture was sequenced on an Illumina MiSeq instrument (Illumina, USA) combined with 50% of control Phi-X control library using a 250 bp paired-end protocol recommended by the manufacturer.
Dna engine tetrad 2
Manufactured by Bio-Rad
Sourced in United States
The DNA Engine Tetrad 2 is a thermal cycler designed for performing polymerase chain reaction (PCR) experiments. It features four independent thermal blocks, allowing for simultaneous processing of multiple samples. The device is capable of precisely controlling temperature and thermal ramp rates to ensure accurate and consistent results.
Automatically generated - may contain errors
4 protocols using dna engine tetrad 2
1
16S rRNA Gene Amplification and Sequencing
2
Genomic DNA Extraction and PCR Amplification
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Total genomic DNA was extracted from young leaves of F2, F3 plants and their parents according to the method of McCouch et al. (1988 (link)) with modifications. Polymerase chain reactions (PCR) were performed in a reaction volume of 20 μl containing 100 ng of template DNA, 0.1 μM each primer, 2.5 mM dNTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01% (w/v) gelatin, and 0.5 U Taq DNA polymerase. PCR amplification was carried out in a DNA Engine Tetrad 2 and Dyad Thermal Cycler (Bio-Rad, USA) using the following reaction conditions: 5 min at 94 °C; followed by 35 cycles of 1 min at 94 °C, 30 s at 56 °C, and 40 s at 72 °C; and 10 min at 72 °C for final extension. PCR products were separated on 2.5% (w/v) agarose gels containing 0.15 μg ml−1 ethidium bromide (EtBr) in 0.5 × TBE buffer.
3
Nanowell Chip Sealing and Centrifugation
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Following all reagent additions, nanowell chips were sealed (Microseal film A, BioRad; pressed on with a pneumatic sealer) and reagents collected at the bottom of the well with a centrifugation step at 3,214 g for 2 min. All chip incubations, with the exception of the cell heat lysis, were carried out on a flatbed thermal cycler (DNA Engine Tetrad 2, Biorad), followed by a centrifugation step for 2 min at 3,214 g.
4
16S rRNA Gene Amplification and Sequencing
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Universal primers 515 F and 806R were used for PCR amplification of the V4 hypervariable region of the 16S rRNA gene according to the procedure of Caporaso et al. [35 (link)]. The 806R primer included a unique 12 bp sequence tag to barcode each sample. The V4 region of the 16S rRNA genes was amplified in 96-well microtitre plates using Phusion High-Fidelity DNA polymerase (Thermo Fisher, USA) and 50 ng template DNA in a total reaction volume of 50 μl. Reactions were run in a DNA Engine Tetrad 2 thermo cycler (Bio-Rad, USA) using the following cycling parameters: 30 s at 98 °C, followed by 25 cycles of 10 s at 98 °C, 15 s at 55 °C, and 15 s at 72 °C, with a final step of 10 min at 72 °C. Negative controls without template were included for each barcoded primer pair. Amplicons were confirmed by gel electrophoresis (2% E-gel) and quantified using the Quant-iT™ PicoGreen® dsDNA assay (Life Technology, USA). Equimolar amounts (100 ng) of each of 278 PCR amplicons were mixed in a single tube. Primers and reaction buffer were removed from the pool of samples using the AMPure kit (Agencourt, USA). The purified amplicon mixture was sequenced on an Illumina MiSeq instrument (Illumina, USA) combined with 50% of control Phi-X control library using a 250 bp paired-end protocol recommended by the manufacturer.
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