The primers used in constructing vectors are listed in Supplemental Table S1 . The full-length coding region of LcERF056 was inserted into the binary vector pH7WG2D and pK7GW1WG2 to generate 35S:LcERF056 and RNAi-LcERF056 constructs, respectively. The obtained plasmid was then introduced into the Agrobacterium tumefaciens strain GV3101. Agrobacterium bearing the plasmids was transformed into Arabidopsis Col-0 plants by using the floral dip method [39 (link)]. The plasmids were introduced into L. corniculatus cotyledon by using Agrobacterium-mediated genetic transformation [40 ]. DNA or RNA was extracted from the transformed plants by using a DNA/RNA extraction kit (TIANGEN, Beijing, China), and PCR or qPCR was performed to identify the transgenic lines. Expression levels of LcERF056 were calculated for different transgenic lines. Primer sequences used for PCR and qPCR are listed in Supplemental Table S2 .
Dna rna extraction kit
Manufactured by Tiangen Biotech
Sourced in China
The DNA/RNA extraction kit is a laboratory equipment designed to efficiently isolate and purify DNA and RNA molecules from a variety of biological samples. The kit contains all the necessary reagents and materials required to perform the extraction process, allowing users to obtain high-quality nucleic acid samples for further analysis and downstream applications.
Automatically generated - may contain errors
4 protocols using dna rna extraction kit
1
Genetic Transformation of Arabidopsis and Lotus
2
Nucleic Acid Extraction and qPCR Analysis
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Nucleic acid extractions from the samples of all treatments (DNA/RNA extraction kit, TianGen, Beijing, China) and cDNA synthesis (Prime ScriptTM RT reagent Kit with gDNA Eraser) were performed according to the manufacturer’s protocol. The concentrations of nucleic acid were determined using the NanoDrop 1000 spectophotometer (NanoDrop Technologies, Wilmington, DE, USA). Primer sequences and PCR product sizes were described in Table 2 . The real time PCR was performed using Applied Biosystems® 7500 Real-Time PCR system, and the PCR was carried out according to the instruction provided by SYBR® Premix Ex Taq™ Kit from TaKaRa. For each experiment, a standard curve was generated using serially diluted plasmid containing N gene or CAP gene.
3
Transcriptome Analysis of Varroa Mite
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Total DNA and RNA were extracted from each mite with a DNA/RNA Extraction Kit (Tiangen, Beijing, China) following the manufacturer’s protocol. To avoid the use of mites that recently drifted to colonies of the other host species in our analysis, mite lineages were confirmed a posteriori by mtDNA amplification and microsatellite DNA analysis [15] .
RNA extracted from five individual mites in each treatment was pooled for transcriptome sequencing. The NEBNext Ultra TM RNA Library Prep Kit (New England Biolabs, Beijing, China) was used to construct the paired-end 150 bp library. RNA-seq was performed with an Illumina HiSeq 1500 system (Novagene, Tianjin, China). At least 6 Gb of 2 × 150 bp sequencing data were generated for each sample.
The filter of low-quality reads was the same as that for genomic sequencing above. TopHat2 [31] (link) was used to align the RNA-seq reads against the V. destructor genome (GenBank Accession No.: BEIS00000000.1). Gene expression was evaluated with RSEM [32] . Differentially expressed genes (DEGs) between individual samples were identified using DEseq2 [33] (link). The expression profile of these DEGs was clustered and a heatmap was produced using the pheatmap package in R software.
RNA extracted from five individual mites in each treatment was pooled for transcriptome sequencing. The NEBNext Ultra TM RNA Library Prep Kit (New England Biolabs, Beijing, China) was used to construct the paired-end 150 bp library. RNA-seq was performed with an Illumina HiSeq 1500 system (Novagene, Tianjin, China). At least 6 Gb of 2 × 150 bp sequencing data were generated for each sample.
The filter of low-quality reads was the same as that for genomic sequencing above. TopHat2 [31] (link) was used to align the RNA-seq reads against the V. destructor genome (GenBank Accession No.: BEIS00000000.1). Gene expression was evaluated with RSEM [32] . Differentially expressed genes (DEGs) between individual samples were identified using DEseq2 [33] (link). The expression profile of these DEGs was clustered and a heatmap was produced using the pheatmap package in R software.
4
LAMP Assay for M. hyopneumoniae Detection
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The amplified p36 PCR product was purified and cloned into the vector pET-28a (+) for the construction of a p36 expression vector [pET-28a (+)-p36]. Purified plasmid DNA, and genomic DNA and cDNA from M. hyopneumoniae and other strains were extracted from bacterial cultures with a commercial DNA/RNA extraction kit (Tiangen), following the manufacturer instructions, for detection of LAMP assay specificity. M. hyopneumoniae genomic DNA from lung tissues was prepared via the boiling method. The resulting supernatant was used as a template for determining the clinical application of the LAMP assay vs real-time PCR.
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