Fenpropathrin, cypermethrin, permethrin, cypermethrin and fenvalerate (purity 98%) were purchased from J&K Scientific Ltd. SBA-15 (pore size 6–11 nm) was bought from XFNANO. T4 DNA ligase, restriction endonuclease and DNA polymerase were provided by Takara. A Bradford protein concentration assay kit was offered by Sigma. All other chemicals were analytically pure commercial products. E. coli DH5α and E. coli BL21 (DE3) were offered by TSINGKE Biological Technology.
E coli dh5α
Manufactured by Tsingke
Sourced in China
E. coli DH5α is a common laboratory strain of the bacterium Escherichia coli. It is widely used as a host for various DNA cloning and recombinant protein expression experiments due to its efficient transformation and growth characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Restriction endonuclease, by Takara Bio (2 mentions)
T4 dna ligase, by Takara Bio (2 mentions)
Cypermethrin, by J&K Scientific (1 mentions)
Dna polymerase, by Takara Bio (1 mentions)
E coli bl21 de3, by Tsingke (1 mentions)
Sba 15, by XFNANO (1 mentions)
Bl21 de3, by Tsingke (1 mentions)
Molecular biology reagents, by Takara Bio (1 mentions)
5 bromo 4 chloro 3 indolyl β d galactopyranoside x gal, by Merck Group (1 mentions)
Isopropyl β d thiogalactopyranoside iptg, by Merck Group (1 mentions)
Escherichia coli bl21 de3, by Tsingke (1 mentions)
Ampicillin amp, by Merck Group (1 mentions)
Kod plus neo polymerase, by Toyobo (1 mentions)
Kod plus neo, by Toyobo (1 mentions)
Pclone007 blunt simple vector, by Tsingke (1 mentions)
Gel extraction kit, by Omega Bio-Tek (1 mentions)
8 protocols using e coli dh5α
1
Purification and Characterization of Pyrethroid Compounds
2
Heterologous Expression of MFS Transporter
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The MFS transporter-encoding gene FKQ53_RS21695 in GI-M202a was amplified using the primer pair MRC-F/R (with Xba I at the 5′-end and Hind III at the 3′-end) and M202 genomic DNA as a template. The promoter of the β-lactamase gene was amplified using the primers AP-F/R (with Hind III at the 5′-end and Xba I at the 3′-end) (Table S1 ) and the pMD18-T vector as a template, and then the above two gene fragments were fused by T4 DNA ligase after Hind III digestion. After digestion by Xba I, the fused fragment was cloned into the pMD18-T vector to obtain recombinant pMD18-Mrc. After verification by DNA sequencing, pMD18-Mrc was transformed into E. coli DH5α (TSINGKE, China) for heterogeneous expression of FKQ53_RS21695, and the recombinant strain was named Mrc-3.
3
Cloning and Expression of xynST7 Gene
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The XynST7-encoding gene (removing the signal peptide sequence) was amplified using the forward primer (5′-ATGGATCCCAGAGCGGCCGCTACTTCGGCAC-3′ with BamH I restriction site) and reverse primer (5′-ATCTCGAGTCAGGTGCGGGTCCAGCGCTG-3′ with Xho I restriction site), and ligated into the pMD 18-T vector. The resulting pMD 18-T-xynST7 was transformed into E. coli DH5α and sequenced by Tsingke Biotechnology Co., Ltd. (Qindao, China). The plasmid pMD 18-T-xynST7 was digested with BamHI and XhoI, and the resulting xynST7 was ligated to the pET-28a (+), which was transformed into E. coli BL21 (DE3). A single colony harboring the plasmid pET-28a-xynST7 was grown at 37 °C for 12 h in 3 mL of Luria–Bertani (LB) medium containing 50 µg/mL kanamycin. This culture was transferred to a fresh LB medium containing kanamycin according to the inoculation amount of 2% (v/v ) and grown at 37 °C with shaking at 200 rpm. When the OD600nm value of the culture reached 0.6, xynST7 expression was induced by adding isopropyl-β-D-thiogalactopyranoside to a final concentration of 1 mM, then cultivation was continued for another 3 h at 37 °C. The xynST7 expression was confirmed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).
4
Biofilm formation and QEN treatment
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The standard MRSA strain (ATCC-33591) and the isolated strains used in this study were preserved in our laboratory. The culture condition of the biofilm was Trypticase soy broth (TSB) medium containing 0.5% glucose (TSB-g). S. aureus was cultured to the logarithmic growth phase, and the bacterial suspension was adjusted to an OD600 of 0.1 (about 1 × 108 CFU/mL). After 100-fold dilution with TSB-g, QEN storage solution (40,960 µg/mL) was added to prepare a suspension of bacteria with different QEN concentrations. E. coli DH5α and BL21 (DE3) were purchased from Tsingke Biotechnology Co., Ltd.
5
Heterologous Expression of G3577_03020
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Gene G3577_03020, encoding a major facilitator superfamily (MFS) transporter, was amplified using primers M3020-F/R (Supplementary Table 1 ) and MP63 genomic DNA as template, and the β-lactamase gene promoter was amplified using primers AP-F/R (Supplementary Table 1 ) and pMD18-T vector as template. These two amplicons were then fused by PCR using primers AP-F and M3020-R (Supplementary Table 1 ), followed by cloning into the pMD18-T vector to obtain the recombinant pMD18-3020. After verification by DNA sequencing, pMD18-3020 was transformed into E. coli DH5α (TSINGKE, China) for heterogeneous expression of G3577_03020, and the recombinant strain was named M3020.
6
Recombinant Protein Expression in E. coli
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Escherichia coli BL21 (DE3) and E. coli DH5α (both from TSINGKE Biological Technology, Guangzhou, China) were used as the expression host and cloning host, respectively. The pET-32a (+) (Novagen) was used for protein expression. T4 DNA Ligases, restriction endonucleases and pUC118/BamH I (BAP) were purchased from TaKaRa (Dalian, China). DNA extraction kit, plasmid extraction kit and His-tag protein puri cation kit (Novagen) were provided by OMEGA (San Diego, CA, USA). Ampicillin (Amp), 5-Bromo-4chloro-3-indolyl-β-D-galactopyranoside (X-Gal), and isopropyl-β-D-thiogalactopyranoside (IPTG) were bought from Sigma. All other chemicals and reagents were produced by RUISHU Biological Technology (Guangzhou, China) unless otherwise stated. Water hyacinth was gathered locally. Molecular biology reagents from TaKaRa were used according to the manufacturer's instructions.
7
Recombinant hSOD1 Expression in Bacillus subtilis
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E.coli DH5α (Tsingke Biotechnology Co., Ltd., Beijing, China) was used for plasmid construction and molecular cloning, and the host strain Bacillus subtilis 1012 (Lab collection) was applied for hSOD1 expression. The E.coli-B.subtilis shuttle plasmid pHT43-His (Miaoling Biological Technology Co., Ltd., Wuhan, China) was constructed as a vector for extracellular expression of hSOD1. The cells were grown at 37°C for 12 h in LB, Super Rich, or 2× YT medium (100 µg/mL Ampicillin for E.coli; 5 µg/mL Chloramphenicol (Cm) for Bacillus subtilis).
8
Cloning and Sequencing of Human SOD1 Gene
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The human RNA was extracted from HepG2 cells using Trizol (Summer Biotechnology Co., Ltd, Beijing, China). cDNAs were synthesized with a Transcriptor rst-strand cDNA synthesis kit (Summer Biotechnology Co., Ltd, Beijing, China). The coding sequence of the hsod1 gene (GenBank accession number, CR541742.1) was ampli ed using a pair of SOD1 primers: 5′-ATGGCGACGAAGGCCGTGTG-3′ (F); 5′-TTGGGCGATCCCAATTACAC-3′ (R).
PCR reaction was performed in 50 µL reaction solution consisting of 2 µL of cDNA, 1 µL each of the forward or reverse primer, 5 µL of dNTP (2 mM), 5 µL of 10× PCR Buffer for KOD-Plus-Neo, 3 µL of MgSO 4 (25 mM), 1 µL of KOD-Plus-Neo Polymerase (Toyobo, Osaka, Japan), and 32 µL of ddH 2 O. The PCR condition was as followed: 94°C for 2 min; 35 cycles of 94°C for 15 s, 55°C for 30 s, and 68°C for 25 s.
The PCR product was puri ed using a Gel Extraction Kit (Omega Bio-tek, Guangzhou, China) according to the manufacturer's instruction. After the fragment was cloned to the pClone007 Blunt Simple Vector (Tsingke Biotechnology Co., Ltd., Beijing, China) and transformed into E.coli DH5α, the positive colony was sequenced (Tsingke Biotechnology Co., Ltd., Beijing, China).
PCR reaction was performed in 50 µL reaction solution consisting of 2 µL of cDNA, 1 µL each of the forward or reverse primer, 5 µL of dNTP (2 mM), 5 µL of 10× PCR Buffer for KOD-Plus-Neo, 3 µL of MgSO 4 (25 mM), 1 µL of KOD-Plus-Neo Polymerase (Toyobo, Osaka, Japan), and 32 µL of ddH 2 O. The PCR condition was as followed: 94°C for 2 min; 35 cycles of 94°C for 15 s, 55°C for 30 s, and 68°C for 25 s.
The PCR product was puri ed using a Gel Extraction Kit (Omega Bio-tek, Guangzhou, China) according to the manufacturer's instruction. After the fragment was cloned to the pClone007 Blunt Simple Vector (Tsingke Biotechnology Co., Ltd., Beijing, China) and transformed into E.coli DH5α, the positive colony was sequenced (Tsingke Biotechnology Co., Ltd., Beijing, China).
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