Primers with restriction enzyme sites BamHI and EcoRI were designed with Primer Premier 5.0 (Table 1 ), and the coding region of PyasOBP2 without the signal peptide was amplified with PCR. The PCR products were ligated into the pMD®19-T vector, transformed into DH5α competent cells (TaKaRa Co., Dalian, China) and then sequenced. The correct pMD®19-T plasmids were digested by restriction enzymes (BamHI and EcoRI) (TaKaRa) for 1–2 h at 37°C, cloned into the digested pET32a vector, and then transformed into DH5α cells. The correct recombinant plasmids were transformed into BL21 (DE3) competent cells (TaKaRa). Single colonies were cultured in liquid LB (supplemented with 100 mg/ml ampicillin) overnight at 37°C.
Dh5α competent cells
Manufactured by Takara Bio
Sourced in China, Japan
DH5α competent cells are a widely used strain of E. coli bacteria that are genetically modified to be highly competent for DNA transformation. They are commonly used in molecular biology laboratories for the cloning and amplification of plasmid DNA.
Automatically generated - may contain errors
Lab products found in correlation
Pmd19 t vector, by Takara Bio (4 mentions)
Pmd18 t vector, by Takara Bio (3 mentions)
5 full race kit, by Takara Bio (2 mentions)
Tks gflex dna polymerase, by Takara Bio (2 mentions)
Pmd19 t simple vector, by Takara Bio (2 mentions)
One step primescript rt pcr kit, by Takara Bio (2 mentions)
Dpni, by Takara Bio (2 mentions)
Ampicillin, by Merck Group (2 mentions)
Pmd18 t vector cloning kit, by Takara Bio (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Generacer, by Thermo Fisher Scientific (2 mentions)
Pgem t easy vector, by Transgene (2 mentions)
3730 dna analyzer, by Thermo Fisher Scientific (2 mentions)
Puc118 hinc 2 bap vector, by Takara Bio (2 mentions)
Bl21 de3 competent cells, by Takara Bio (1 mentions)
Dh5α cells, by Takara Bio (1 mentions)
Dna purification kit, by Tiangen Biotech (1 mentions)
Trelief sosoo cloning kit, by Tsingke (1 mentions)
Gel extraction kit, by Omega Bio-Tek (1 mentions)
46 protocols using dh5α competent cells
1
Cloning and Expression of PyasOBP2
2
Cloning and Validating Promoter SNPs
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The DNA sequence (length ranges from ~300 to 700 bp) containing the test SNP was amplified using primers (Supplementary Table 10 ) linked with homologous arms (which were identical with the sequence (located at the multiple clone sites) of pGL3-promoter vectors) (Supplementary Fig. 12 ) and PCR products were purified with DNA Purification Kit (DP209, TIANGEN). We digested pGL3-Promoter vector (E1761, Promega) and pGL4.11 vector (E6661, Promega) with KpnI (FD0524, FastDigest) and XhoI (FD0694, FastDigest), and the digestion products were purified with DNA Purification Kit. Then the purified DNA fragments containing the test SNP were inserted into the pGL3-Promoter vector using the TreliefTM SoSoo Cloning Kit (TSV-S1, TSINGKE). As SNP rs2270363 is located in promoter region, we inserted the DNA fragments containing rs2270363 into the pGL4.11, which is a basic vector with no promoter. The ligated vectors were then used to transform DH5α competent cells (Takara). LB plates (with the selectable marker, ampicillin) were used to select the transformed cells and recombinant plasmids were extracted from the transformed cells grown from single colony. PCR-mediated point mutation technique was used to generate the DNA fragments containing the alternative allele of this test SNP. All sequences of the inserted DNA fragments were verified using Sanger sequencing.
3
Knockdown of ROCKII in Mouse Microglia
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A lentivirus-based shRNA system was constructed by NeuronBiotech Co., Ltd. (Shanghai, China). Briefly, DH5α competent cells (Takara Bio, Inc., Otsu, Japan) were used to produce the lentivirus vector, pLKD.CMV.GFP.U6, with U6 promoter as the transcriptional start site for shRNA. A 21-bp sequence containing a stem loop sequence was inserted into the vector to produce a valid shRNA that would interfere with ROCKII at the mRNA level (NM_009072.2). Five candidate lentiviruses targeting five unique sequences were constructed and an empty lentivirus vector served as a control. Lentiviruses were transfected into the mouse BV2 microglia cell line for screening. When cell density reached ~30%, lentiviruses were added into the culture medium with a multiplicity of infection of 20 for 24 h. Protein levels and enzyme activity of ROCKII in the culture medium were measured to evaluate the shRNA interference systems once at 5–8 days following transfection.
4
Sequencing Avian Coronavirus Genomes
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Pairs of primers targeting the S1 gene and 17 fragments of the complete IBV genome (Supplementary Table 1) were designed to amplify, clone, and sequence the genomes of isolates using Oligo 7. The genomic sequence of the GDTS13 strain and sequences of the S1 gene of other isolates were verified by RT-PCR using a PrimeScript One Step RT-PCR Kit Ver. 2 (Takara). Target products obtained by RT-PCR were purified and recovered using a Gel Extraction Kit (Omega Bio-tek, Norcross, GA, USA). The RT-PCR products were ligated into the pMD19-T vector (Takara) and used to transform DH5α competent cells (Tiangen, Beijing, China). Plasmids positive for these products were sequenced by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China).
5
Cloning and Characterization of TaGli-γ-2.1 Gene
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TaGli-γ-2.1 partial sequences were used for BLASTN search in NCBI database. Three sequences (GenBank accession JX081265, JX081266 and JX081267) were identified in wheat and related species [24] (link). According to the conserved open reading frame (ORF) region, degenerate primers were designed using Primer3 software [25] (link) to clone TaGli-γ-2.1 coding regions (Table S1). Genomic DNA and cDNA from Zhengmai 004 were used as templates for PCR amplification.
According to TaGli-γ-2.1 sequences, primers were designed to clone 3′ downstream and 5′ upstream sequences, respectively. Total RNA was extracted to synthesize cDNA following the protocol of 3′- and 5′-Full RACE Kit (AK1501, TaKaRa, Shiga, Japan), respectively. The 3′ and 5′ Untranslated Region (UTR) were obtained by Nested-PCR. The promoter sequence of TaGli-γ-2.1 was cloned with the promoter specific primers designed based on the wheat reference genome sequence (Table S1) using Phusion® High-Fidelity DNA Polymerase (M0530, New England Biolabs, Massachusetts, USA). The PCR products were cloned into the pEASY-Blunt3 vector (TransGen Biotech, Beijing, China), and were transformed into DH5α competent cells (TaKaRa, Shiga, Japan).
According to TaGli-γ-2.1 sequences, primers were designed to clone 3′ downstream and 5′ upstream sequences, respectively. Total RNA was extracted to synthesize cDNA following the protocol of 3′- and 5′-Full RACE Kit (AK1501, TaKaRa, Shiga, Japan), respectively. The 3′ and 5′ Untranslated Region (UTR) were obtained by Nested-PCR. The promoter sequence of TaGli-γ-2.1 was cloned with the promoter specific primers designed based on the wheat reference genome sequence (Table S1) using Phusion® High-Fidelity DNA Polymerase (M0530, New England Biolabs, Massachusetts, USA). The PCR products were cloned into the pEASY-Blunt3 vector (TransGen Biotech, Beijing, China), and were transformed into DH5α competent cells (TaKaRa, Shiga, Japan).
6
Cloning and Overexpression of LrDFR1 from L. radiata
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Cloning of LrDFR1 was based on putative ORFs of unigenes from the RNA-seq database. Primers (Supplementary Table 1 ) were synthesized for ORF sequence amplification using Tks Gflex™ DNA Polymerase (Takara, Dalian, China) from L. radiata petal cDNA. Reaction conditions were: 5 min of 95°C, 35 cycles for 30 s at 94°C, 30 s at 60°C, 1 min at 72°C, with extension at 72°C for 10 min. PCR products were cloned into pMD19-T simple vectors (Takara, Dalian, China). Afterward, those T-vectors were transferred into DH5α competent cells (Takara, Dalian, China) for amplification. The overexpression vectors of LrDFR1 were established by linking their ORFs into a linear plant transformation vector, pBinGFP4, using the One Step Cloning Kit (Vazyme, Nanjing, China). Then the 35S:LrDFR1 recombinant vectors were transformed into Agrobacterium tumefaciens EHA105 competent cells.
7
Molecular Detection of Swine Viral Pathogens
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RNA/DNA was extracted from a clinical PRCoV-positive specimen or a PRRSV, PRV, and SIV vaccine solution and was used as a template to amplify PRCoV, PRRSV, SIV, and PRV gene fragments via PCR using the primers in Table 1 with a One Step PrimeScript™ RT-PCR Kit (Perfect Real Time) (TaKaRa, Dalian, China). The PCR products were purified using a MiniBEST DNA Fragment Purification Kit Ver.4.0 (TaKaRa, Dalian, China), cloned into a pMD18-T vector (TaKaRa, Dalian, China), and transformed into DH5α competent cells (TaKaRa, Dalian, China). Positive clones were cultured at 37 °C overnight for 20–24 h, and plasmid constructs were extracted using a MiniBEST Plasmid Extraction Kit Ver.5.0 (TaKaRa, Dalian, China). The four standard plasmid constructs were named p-PRCoV, p-PRRSV, p-SIV, and p-PRV. The OD260/OD280 nm values of the standard plasmid constructs were measured, and their concentrations were determined using the following formula:
8
Bisulfite Genomic Sequencing of KYSE70 Cell Line
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Genomic DNA extracted from the KYSE70 cell line using the Takara MiniBEST Universal Genomic DNA Extraction kit (Takara), was modified by sodium bisulfite using the EpiTect Bisulfite kit (Qiagen, Germany) according to the manufacturer’s instructions. Methylation status was analyzed by bisulfite genomic sequencing (BSP) of the CpG islands. The region was amplified using the primers shown in Table I . Amplified products were cloned into pMD-18T simple vector (Takara), transformed into DH5α competent cells (Takara), and plated under ampicilin selection. Five independent clones were sequenced.
9
Safflower Genetic Transformation Protocol
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Safflower seeds were provided by Fuyu seeds company, China. Escherichia coli BL21 (DE3), Escherichia coli TransT1, DH5α competent cells, and A. tumefacien strain EHA105 were purchased from Takara Biotechnology Company Beijing, China. Plant overexpression vector (pBASTA) and pCAMBIA1302-GFP-35S were purchased from TransGen.
Biotech, Beijing and preserved in our laboratory (Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development at Jilin Agricultural University, Jilin, China) until next use. Restriction enzymes, GºTaq DNA polymerase and DNA ligases were purchased from Takara Biotechnology Company Beijing, China.
Biotech, Beijing and preserved in our laboratory (Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development at Jilin Agricultural University, Jilin, China) until next use. Restriction enzymes, GºTaq DNA polymerase and DNA ligases were purchased from Takara Biotechnology Company Beijing, China.
10
Cloning and Sequencing ITS1 Sequences
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The purified PCR products were cloned in Escherichia coli and connected with pMD18-T (TakaRa, Dalian, China) and then transferred into DH5α Competent Cells (TaKaRa, Dalian, China). Positive clones were screened by bacterial PCR and sent to Shenggong Corporation (Shanghai, China) for sequencing. The plasmid DNAs were extracted using the Plasmid Kit (Omega, Guangzhou, China). These plasmids containing ITS1 sequence of A. ceylanicum and A. caninum were named AceP and AcaP, respectively. The A260/A280 value and concentration of plasmids (1 μL) were measured using ultra-micro-spectrophotometer, where A260/A280 value and optimal concentration should be 1.8~2.0 and 50 ng/μL. These plasmids were stored at −20°C for use.
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