To generate the TaSTT3b overexpression vector, the full ORF sequence of TaSTT3b‐2B plus a 6 × His epitope tag was amplified using the primers containing the BamH I and Sac I restriction sites and then inserted into the monocot transformation vector pWMB122 (Wang et al., 2017 (link)). In the pWMB122‐TaSTT3b‐2B vector (Figure S10 ), the TaSTT3b‐2B‐His fusion protein is driven by the maize ubiquitin (Ubi) promoter and terminated by the 3’–non‐transcribed region of the Agrobacterium tumefaciens nopaline synthase (Tnos) gene. To generate TaSTT3b transgenic wheat plants, the pWMB122‐TaSTT3b‐2B vector was introduced into hexaploid wheat (cv. Zhoumai18) via Agrobacterium‐mediated transformation. Herbicide spraying and PCR were used to select the positive transgenic plants. Herbicide spraying was performed as described by Wang et al. (2017 (link)). For the PCR experiment, reactions containing about 200 ng genomic DNA, 10 μL 2×Taq MasterMix (TransGen Biotech, China), and 1 μL of each primer (10 mμ ) were prepared. The PCR products were resolved on a 1.5% agarose gel and visualized after ethidium bromide staining. The primers used in this assay are listed in Table S6 .
Taq mastermix
Manufactured by Transgene
Sourced in China
2×Taq MasterMix is a pre-mixed solution containing Taq DNA polymerase, buffer, and dNTPs, ready-to-use for PCR applications. It is designed to simplify the PCR setup process and provide consistent results.
Automatically generated - may contain errors
3 protocols using taq mastermix
1
Generation of TaSTT3b Overexpression Wheat
2
Bacterial 16S rRNA Gene Sequencing
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DNA was then extracted using the HiPure Stool DNA Kit (Magen, Guangzhou, China), and their quantity and quality were determined using the Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and by agarose gel electrophoresis. Subsequently, the V4 region of the bacterial 16S rRNA gene was amplified in 50 μL reaction mixtures using 2× Taq master Mix (TransGen Biotech Co., Ltd., Beijing, China) with 5 μM each of the primers 515F and 806R.
According to the operation manual (Gene Denovo Biotechnology Co., Ltd., Guangzhou, China), the thermal cycling conditions were as follows: initial denaturation at 95 °C for 2 min followed by 27 cycles at 98 °C for 10 s, 62 °C for 30 s, and 62 °C for 30 s, and a final extension at 68 °C for 10 min. The products were recovered and quantified with a QuantiFluor™ fluorometer (Promega, Madison, WI, USA). The PCR amplicons were sequenced on the Illumina HiSeq2500 platform (Novogene Bioinformatics Technology Co. Ltd., Tianjin, China). The raw data were deposited in the Sequence Read Archive database of NCBI (BioProject: PRJNA766330).
According to the operation manual (Gene Denovo Biotechnology Co., Ltd., Guangzhou, China), the thermal cycling conditions were as follows: initial denaturation at 95 °C for 2 min followed by 27 cycles at 98 °C for 10 s, 62 °C for 30 s, and 62 °C for 30 s, and a final extension at 68 °C for 10 min. The products were recovered and quantified with a QuantiFluor™ fluorometer (Promega, Madison, WI, USA). The PCR amplicons were sequenced on the Illumina HiSeq2500 platform (Novogene Bioinformatics Technology Co. Ltd., Tianjin, China). The raw data were deposited in the Sequence Read Archive database of NCBI (BioProject: PRJNA766330).
3
Monitoring TaCOMT-3D Transgene in Wheat
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The presence of the TaCOMT-3D-overexpressing transgene in the transformed wheat plants was monitored by PCR using the specific primers, TaCOMT-3D-F (5′-GAGAGGTACGAGAGGGAGTT-3′, located in TaCOMT-3D coding sequence) and Nos-R (5′-TAAATGTATAATTGCGGGAC-3′, located in Tnos of the transformation vector). PCR was performed in a 25 μl volume containing ~200 ng genomic DNA, 12.5 μl 2 × Taq MasterMix (TransGen Biotech, China), 1 μl each primer (10 mΜ). The amplified product (318-bp size) specific to the introduced TaCOMT-3D-Tnos chimera was resolved on a 1.5% agarose gel and visualized by ethidium bromide staining.
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