Dh5α cells

Manufactured by Transgene

Sourced in China

DH5α cells are a strain of E. coli bacterial cells commonly used in molecular biology laboratories. They are designed for the efficient transformation and propagation of plasmid DNA. DH5α cells exhibit high transformation efficiency, making them suitable for cloning and recombinant DNA experiments.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Pcdna3, by Addgene (1 mentions) Plko 1 puro, by Addgene (1 mentions) Dna gel extraction kit, by Tiangen Biotech (1 mentions) Pmd19 t vector, by Takara Bio (1 mentions) Bigdye terminator v3, by Thermo Fisher Scientific (1 mentions) Peasy t1 simple cloning vector, by Transgene (1 mentions) Agarose gel recovery kit, by Tiangen Biotech (1 mentions)

Spelling variants of this product

Escherichia coli DH5α competent cells (10 citations) Escherichia coli DH5α cells (9 citations) DH5α competent cells (7 citations) E. coli DH5α competent cells (7 citations) E. coli DH5α cells (5 citations) E. coli DH5α and BL21 (DE3) competent cells (2 citations)

5 protocols using dh5α cells

TRIM52 knockdown in HepG2 cells

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The pcDNA3.1(+) was obtained from Addgene (Cambridge, MA, USA). HBx-expressing vector (HBx-pcDNA3.1) was constructed using the primers as follows:
HBx-pcDNA3.1 was transiently transfected into HepG2 cells using Lipofectamine 2000 (Invitrogen). The transfection efficiency was confirmed by qRT-PCR and Western blot analysis.
A short hairpin RNA (shRNA) lentiviral vector targeting position 670–692 (GGGCATGTGCTTTAAACAC) of human TRIM52 gene was used to suppress the expression of TRIM52. We designed and synthesized the human TRIM52-shRNA. Then, the annealed TRIM52-shRNA was cloned into linearized pLKO.1-puro (Addgene) and transformed into DH5α cells (TransGen Biotech, Beijing, China). Positive clones were collected for PCR identification. The primer sequences were as follows: forward: 5′-CAAGGTCGGGCAGGAAGAG-3′; reverse: 5′-TAGAAGGCACAGTCGAGG-3′. The endotoxin-free recombinant expression vector was extracted and identified by sequencing. Finally, the recombinant plasmids pLKO.1-shTRIM52, psPAX2, and pMD2G were co-transfected into 293T cells. The supernatant containing viral particles was collected. HepG2.2.15 cells were transfected with recombinant TRIM52-shRNA lentiviral vector or negative control lentiviral vector, respectively. The silencing of TRIM52 was confirmed by qRT-PCR and Western blot analysis.

Zhang Y., Wu S.S., Chen X.H., Tang Z.H., Yu Y.S, & Zang G.Q. (2017). Tripartite Motif Containing 52 (TRIM52) Promotes Cell Proliferation in Hepatitis B Virus-Associated Hepatocellular Carcinoma. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 23, 5202-5210.

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DNA Extraction and PCR Amplification

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A rectangular gel slice containing a target band was excised and soaked in 10 μL sterilized double-distilled water at room temperature for 10 min and then in a water bath at 80°C for 15 min. After a short centrifugation, the liquid was transferred to a clean tube. The extracted DNA was used directly as the template for PCR [9 (link)]. The PCR conditions were identical to those used for DDRT-PCR. The reamplified products were visualized on 1% agarose gels and recovered using a DNA gel extraction kit (Tiangen, Beijing, China). All screened fragments were subcloned into vector pMD19-T (Takara Bio Inc., Otsu, Shiga, Japan), and recombinant plasmids were used to transform competent DH5α cells (Transgene, Beijing, China). Blue/white screening and PCR were performed to differentiate recombinant clones. Invitrogen (Beijing, China) sequenced the inserted DNA fragments of the positive clones.

Dang Z.H., Qi Q., Zhang H.R., Li H.Y., Wu S.B, & Wang Y.C. (2014). Identification of Salt-Stress-Induced Genes from the RNA-Seq Data of Reaumuria trigyna Using Differential-Display Reverse Transcription PCR. International Journal of Genomics, 2014, 381501.

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Bisulfite Sequencing of Validated Gene

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DNA (1–500 ng) of validated gene was bisulfite-converted using the EpiTect® Bisulfite kit. The PCR-amplified DNA was cloned into vector pEASY-T1 Simple Cloning Vector (TransGen Biotech, China) and transformed into DH5α cells (TransGen Biotech, China), sequenced by terminal labeling using BigDye Terminator v3.1 (Applied Biosystems, http://www.appliedbiosystems.com/). The sequencing information was analyzed using analysis with BiQ Analyzer. The target sequence was set as the subject and the raw reads as the query and applied blastn to align the reads to the target sequences (E-value < 1e-3, matched sequences ≥30). 100% of the reads were successfully mapped to the target regions of gene.

Lu Y.C., Feng S.J., Zhang J.J., Luo F., Zhang S, & Yang H. (2016). Genome-wide identification of DNA methylation provides insights into the association of gene expression in rice exposed to pesticide atrazine. Scientific Reports, 6, 18985.

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Manipulation of ARHGAP24 Expression in MDA-MB-231 Cells

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To investigate the regulatory function of ARHGAP24 in MDA-MB-231 cells, we constructed the lentiviral vectors to reduce or increase ARHGAP24 expression. We designed the RNAi (RNA interference) sequence targeting position 727–749 (GATCGGATGACAGCAAATC) of human ARHGAP24 gene and synthesized the short hairpin RNA (shRNA). Then, ARHGAP24-shRNA was integrated into pLKO.1-puro (Addgen, Cambridge, MA, USA) and transferred into DH5α cells (TransGen, Beijing, China). The colonies were identified by PCR. Afterwards, pLKO.1-shARHGAP24, psPAX2, and pMD2G plasmids were extracted from the bacteria solution by E.Z.N.A.^® Endo-free Plasmid Mini Kit I (Omega, USA) and cotransfected into 293T cells for 4–6 h. After 48-h transfection, the supernatant was collected for viral transduction in MDA-MB-231 cells.
Similarly, ARHGAP24 ectopic expression lentiviral vector was constructed by integrating the coding sequence (CDS) of ARHGAP24 into pLVX-Puro. The primers used to synthesize the CDS were as follows: forward: 5′-GCGAATTCATGGAGGAGAACAATGACT (EcoR I); reverse: 5′-CGGGATCCCTGAATCCATATTGTGTTT (BamH I) (underscoring indicates the restriction enzyme cutting site). Virus particles were produced by transfecting 293T cells with ARHGAP24-pLVX-Puro together with viral packaging vectors (psPAX2, pMD2G) as described above, and then MDA-MB-231 cells were infected with virus-containing supernatants.

Dai X., Geng F., Dai J., Li M, & Liu M. (2018). Rho GTPase Activating Protein 24 (ARHGAP24) Regulates the Anti-Cancer Activity of Sorafenib Against Breast Cancer MDA-MB-231 Cells via the Signal Transducer and Activator of Transcription 3 (STAT3) Signaling Pathway. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 24, 8669-8677.

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Plasmid Construction and Verification

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The PCR product was subjected to electrophoresis gel, and the target fragment was recovered using an agarose gel recovery kit (Tiangen, Beijing, China). The recovered product was ligated to the pGL3-Basic vector; ligation system (10 µL): 4 µL of recovered PCR product, 1 µL of vector, 5 µL of solution I. The ligation reaction was carried out at 4 °C for over 12 h. A volume of 5 µL of the ligated product was added to 100 µL DH5α cells (TransGen, Beijing, China); after heat-shock in a 42 °C water bath for 90 s, the centrifuge tube was quickly transferred to an ice bath for 1–2 min. Liquid LB medium (395 µL) was added, and the mix was shaken at 200 rpm at 37 °C for 90 min. Then, 200 µL of LB medium containing DH5α was added to AMP medium at 37 °C and cultured for 12–16 h. Single colonies were selected for culture and plasmid purification. Colony screening was performed by polymerase chain reaction (PCR) and double enzyme digestion (NheI/XhoI) experiments. The positive plasmid was sent to the Shanghai Sangon Bioengineering Company for sequencing. Plasmids with the correct sequence were amplified in large quantities and stored at −20 °C for later use.

Sui Z., Zhang Y., Zhang Z., Wang C., Li X., Xing F, & Chu M. (2023). Analysis of Lin28B Promoter Activity and Screening of Related Transcription Factors in Dolang Sheep. Genes, 14(5), 1049.

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