Dh5α competent cells

Manufactured by Transgene

Sourced in China

DH5α competent cells are a strain of Escherichia coli that are commonly used in molecular biology for the transformation of plasmid DNA. They are capable of taking up and maintaining recombinant DNA, making them a useful tool for DNA cloning and protein expression experiments.

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Lab products found in correlation

Trizol reagent, by Takara Bio (2 mentions) Pmd19 t vector, by Takara Bio (2 mentions) Premix ex taq hot start version, by Takara Bio (1 mentions) Primescript rt reagent kit, by Takara Bio (1 mentions) Cdna synthesis kit, by Takara Bio (1 mentions) Kod plus mutagenesis kit, by Toyobo (1 mentions) Rneasy micro purification kit, by Qiagen (1 mentions) 3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions) Ta vector, by Takara Bio (1 mentions) Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions) Pmd18 t vector, by Takara Bio (1 mentions) Kod dna polymerase, by Toyobo (1 mentions) Xbai, by Thermo Fisher Scientific (1 mentions) T4 ligase, by Transgene (1 mentions) Pmd19 t simple vector, by Takara Bio (1 mentions) Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions) Primestar gxl dna polymerase, by Takara Bio (1 mentions)

Spelling variants of this product

Escherichia coli DH5α competent cells (10 citations) E. coli DH5α competent cells (7 citations) DH5α cells (5 citations) E. coli DH5α and BL21 (DE3) competent cells (2 citations)

7 protocols using dh5α competent cells

Molecular Characterization of LvHSSP Gene

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The total RNA was extracted by TRIzol reagent (Takara, Kyoto, Japan) and the cDNA template was synthesized using the PrimeScript RT Reagent Kit (Takara, Kyoto, Japan) with random primers according to the manufacturer’s protocols. Two specific primers LvHSSP-1F and LvHSSP-1R (Table S1) were designed to amplify and validate the nucleotide sequence of LvHSSP from the genome and transcriptome data [22 (link)]. Premix Ex Taq Hot Start version (TaKaRa, Kyoto, Japan) was used to amplify the gene. After the quality was assessed by electrophoresis on 1% agarose gel, the specific product was purified using the Gel Extraction Kit (Omega, Norcross, GA, USA), cloned into the pMD19-T vector (TaKaRa, Kyoto, Japan) and transformed into DH5α competent cells (TransGen Biotech, Beijing, China) for sequencing.
The complete ORF and amino acid sequence of LvHSSP was deduced using ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 2 June 2022). Conserved protein domains were predicted with SMART (http://smart.embl-heidelberg.de/, accessed on 2 June 2022) and InterPro (http://www.ebi.ac.uk/interpro/, accessed on 2 June 2022). The theoretical isoelectric point (pI) and molecular weight (Mw) were calculated using ExPASy (https://web.expasy.org/compute_pi/, accessed on 7 June 2022).

Sun M., Li S., Yu Y., Zhang X, & Li F. (2023). A Novel Hemocyte-Specific Small Protein Participates in White Spot Syndrome Virus Infection via Binding to Viral Envelope Protein. Viruses, 15(1), 227.

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Molecular Cloning of FBXO34 and Plasmid Generation

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RNeasy micro purification kit (Qiagen) was used to extract total RNA from 200 mouse GV oocytes as a sample, and then cDNA synthesis kit (Takara) and poly(dT) primers were used to generate the first strand cDNA. The full length of FBXO34 CDS was amplified by the polymerase chain reaction (PCR) method. The PCR products and Myc tag plasmid were, respectively, digested using FseI and AscI (New England Biolabs, Inc.), and then FBXO34 CDS and Myc plasmid were linked. The fusion plasmid was transfected into DH5α competent cells (Transgene Biotech). KOD-Plus-Mutagenesis Kit (Toyobo) was used to make a synonymous mutation of FBXO34 plasmid. CCNB1-GFP plasmid, MAP4-eGFP plasmid, and H2B-mCherry plasmid were acquired from plasmid bank in our laboratory.

Zhao B.W., Sun S.M., Xu K., Li Y.Y., Lei W.L., Li L., Liu S.L., Ouyang Y.C., Sun Q.Y, & Wang Z.B. (2021). FBXO34 Regulates the G2/M Transition and Anaphase Entry in Meiotic Oocytes. Frontiers in Cell and Developmental Biology, 9, 647103.

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Monoclonal Screening and Sequencing

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The recovered PCR products were linked with TA vectors (Takara, Bao Bioengineering Co., Ltd., China, Dalian), and DH5α competent cells (TransGen Biotech Co., Ltd., Beijing, China) were added for monoclonal screening, and 10 clones were selected for sequencing. The sequencing kit used in this experiment was the BigDye^® Terminator v3.1Cycle Sequencing Kit (Thermo Fisher, Waltham, MA, USA), and the sequencing instrument was a 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA).

Zhang J., Zhang X.Q., Ling X.Z., Zhao X.H., Zhou K.Z., Wang J.Y, & Zhang G.X. (2022). Prediction of the Effect of Methylation in the Promoter Region of ZP2 Gene on Egg Production in Jinghai Yellow Chickens. Veterinary Sciences, 9(10), 570.

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Storing and Handling Bacterial Strains

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Staphylococcus aureus subsp. aureus Rosenbach (ATCC^® 25923™) and Enterococcus faecium HDRsEF1 were stored in the Veterinary Microbiology and Immunology Laboratory of Huazhong Agricultural University (Wuhan, China). DH5α™ competent cells were obtained from TransGen Biotech (Beijing, China) and Pmd18-T Vector was purchased from Takara (Dalian, China).

Wu X., Wu B., Li Y., Jin X, & Wang X. (2021). Identification and safety assessment of Enterococcus thailandicus TC1 isolated from healthy pigs. PLoS ONE, 16(7), e0254081.

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Cloning and Overexpression of FcMYB Genes

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FcCHS1, FcCHI1, FcDFR1, FcMYB21, and FcMYB123 were cloned based on the putative ORFs of fig unigenes from our RNA-seq data. The specific primer pairs (Table S8) were designed to amplify the ORF sequences using KOD DNA polymerase (TOYOBO, Osaka, Japan) from the cDNA of red fig peels. The reaction conditions were as follows: 96 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min, with 10 min extension at 72 °C. The PCR products were cloned into the pMD19-T simple vectors (TaKaRa, Shiga, Japan). Afterwards, those T-vectors were transferred into DH5α competent cells (Transgen Biotech, Beijing, China) for amplification, and the products were sequenced by GENEWIZ (New Jersey, USA).
The overexpression vectors of FcMYB21 and FcMYB123 were constructed via linking their ORFs into the linearized plant transformation vector pBI121 using fast-digest restriction enzymes of XbaI and BamHI (Thermo Scientific, Waltham, USA) and T4 ligase (Transgen Biotech, Beijing, China). Then the 35S::FcMYB21 and 35S::FcMYB123 recombinant vectors were transformed into Agrobacterium tumefaciens EHA105 competent cells, respectively.

Li J., An Y, & Wang L. (2020). Transcriptomic Analysis of Ficus carica Peels with a Focus on the Key Genes for Anthocyanin Biosynthesis. International Journal of Molecular Sciences, 21(4), 1245.

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Amplification and Cloning of OY-TES-1 ORF

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The open reading frame (ORF) of OY-TES-1 was amplified from the cDNA of tumor tissues using PCR with specific primers as follows: Sense, 5′-GCGGCGGATCTTCTCCGGCCATG-3′ and antisense, 5′-ACGGGATCCTTATCAGTTGGGCTGGGGTGT-3′. A total of 35 PCR amplification cycles were performed, each consisting of denaturation at 98°C for 10 sec, followed by annealing at 63°C for 15 sec and extension at 72°C for 2 min. The final extension step was performed at 72°C for 10 min. PCR products were purified and ligated into pMD8-T vectors (Takara Biotechnology Co.), which were transformed into DH5α competent cells (Beijing TransGen Biotech Co., Ltd., Beijing, China) (24 ). The transformed cells were smeared on LB-ampicillin agar plates containing X-gal. White colonies were screened and then inoculated into 5 ml bacterial culture medium overnight. Plasimid was extracted by EZ Spine Column Plasimid Mini-Preps kit (Sangon Biotechnology Co., Shanghai, China) and verified by PCR, as previously described (21 (link)). Clones with the correct insertion were identified via Sanger sequencing in 3730XL DNA Analyzer (Sino Genomax Co., Ltd., Beijing, China).

Li X., Yan J., Fan R., Luo B., Zhang Q., Lin Y., Zhou S., Luo G., Xie X, & Xiao S. (2017). Serum immunoreactivity of cancer/testis antigen OY-TES-1 and its tissues expression in glioma. Oncology Letters, 13(5), 3080-3086.

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Molecular cloning and bioinformatic analysis of TRIM9-1 gene

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The total RNA was extracted by TRIzol reagent (Takara, Tokyo, Japan), and the cDNA template was synthesized using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocols. Two specific primers LvTRIM9-1-1F and LvTRIM9-1-1R (Table S1) were designed to amplify and validate the sequence of LvTRIM9-1 from the genome and transcriptome data (31 (link)). PrimeStar GXL DNA Polymerase (Takara, Japan) was used to amplify the gene. After the quality was assessed by electrophoresis on 1% agarose gel, the specific product was purified using Gel Extraction Kit (Omega, Norcross, GA, USA), cloned into the pMD19-T vector (TaKaRa, Japan), and transformed into DH5α competent cells (TransGen, China) for sequencing.
The complete ORF and amino acid sequence of LvTRIM9-1 was deduced using ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/). Conserved protein domains were predicted with SMART (http://smart.embl-heidelberg.de/). Different TRIM protein sequences (Table S2) were obtained from the UniProtKB/Swiss-prot and NCBI database. Multiple-sequence alignment and phylogenic analysis were performed using the neighbor-joining (NJ) method by ClustalW and MEGA 6.

Sun M., Li S., Jin S., Li X., Xiang J, & Li F. (2022). A Novel TRIM9 Protein Promotes NF-κB Activation Through Interacting With LvIMD in Shrimp During WSSV Infection. Frontiers in Immunology, 13, 819881.

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