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E coli dh5α

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E. coli DH5α is a common laboratory strain of the bacterium Escherichia coli. It is widely used in molecular biology and genetic engineering applications for the cloning and propagation of plasmid DNA.

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Lab products found in correlation

Lb medium, by Merck Group (2 mentions) T7 phage, by Merck Group (2 mentions) 0.45 μm membrane, by Merck Group (2 mentions) Escherichia coli dh5α, by Takara Bio (2 mentions) B subtilis, by Merck Group (1 mentions) E coli dh5α, by Thermo Fisher Scientific (1 mentions) Lb broth, by Merck Group (1 mentions) Brucella broth, by BD (1 mentions) Ampicillin, by Merck Group (1 mentions) S aureus atcc 12600, by Merck Group (1 mentions)

8 protocols using e coli dh5α

1

Bacterial Killing Assay for Bone Marrow Neutrophils

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One milliliter of BMNs (2 × 106 cells/ml) was cultured in RPMI 1640
medium containing 10% FBS in a 24-well flat-bottom plate pre-coated with
poly-L-lysine for 1 h at 37°C. Bacterial killing assays were
performed as described previously.31 (link) 4 × 107 E. coli (DH5α; Sigma-Aldrich, St Louis, MO) were
added in the well containing 2 × 106 of BMNs. After centrifuging the
24-well plate at 600 g for 2 min and incubating
the plate at 37°C for 20 min for E. coli uptake,
cells were washed slightly with PBS three times and lysed with 500 µl 0.1%
Triton X-100 for 5 min. Cell lysates were plated on Luria–Bertani agar plates
and cultured overnight (16 h) at 37°C to determine phagocytic capability by
counting the number of colony forming unit (CFU1). To examine the
bacterial killing capability of BMNs, 2 × 106 BMNs/ml were incubated
with E. coli for 20 min. Cells were washed
slightly with PBS three times and cultured at 37°C for an additional 15 min.
BMNs were lysed with 500 µl 0.1% Triton X-100 for 5 min. Cell lysates were
plated on Luria–Bertani agar plates and cultured overnight at 37°C to determine
bacterial killing capability by counting the number of bacterial colonies
(CFU2). The percent of bacterial killing was calculated as 100 ×
(1 – CFU2/CFU1).
Qian X., Zhao H., Chen X, & Li J. (2018). Disruption of transient receptor potential melastatin 2 decreases elastase release and bacterial clearance in neutrophils. Innate Immunity, 24(2), 122-130.
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2

Isolation and Characterization of Lactobacilli

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All the lactobacilli used in this study were originally isolated from vaginal swabs of healthy women using culture-based methods (27 ). The present study was approved by the Ethical Committee of the Second People's Hospital of Yunnan Province. All volunteers provided written consent and were informed of the purposes of the study. Six strains were identified as L. crispatus, L. gasseri, L. jensenii, L. fermentum, L. delbrueckii and L. johnsonii (detailed in the Results and Table S1). All strains were stored at −80°C in MRS media containing 30% glycerol (v/v) and plated on agarose containing MRS media (Hopebio, Qingdao, China) at 37°C for 96 h before individual colonies were used for routine cultures in liquid MRS medium (27 ).
For the indicated experiments, bacterial cells were exposed to 60Co radiation (10 KGy) for 10 h, and inactivation was assessed by inoculating MRS plates. E. coli JM109, E. coli DH5α, and B. subtilis purchased from Sigma-Aldrich (Gallen, Switzerland) were used as controls.
Song J., Lang F., Zhao N., Guo Y, & Zhang H. (2018). Vaginal Lactobacilli Induce Differentiation of Monocytic Precursors Toward Langerhans-like Cells: in Vitro Evidence. Frontiers in Immunology, 9, 2437.
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3

Microbial Expression and Antimicrobial Evaluation

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E. coli DH5α was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used to prepare expression plasmids. The expression host strain T. reesei Tu6 ATCC MYA-256 was obtained from the American Type Culture Collection (ATCC). The expression vector of T. reesei Tu6, constructed with reference to a previous report [33 (link)] and named PCBHG, was deposited in our laboratory. Of the tested bacterial strains E. coli ATCC 25922, S. derby ATCC 13076, and C. perfringens CVCC 2032, the first two were obtained from the ATCC, and the third from the China Veterinary Culture Collection Center (Beijing, China). The three bacteria were used to measure the antimicrobial activity of the expressed peptide BMGlv2. All chemicals and reagents used in this study were purchased from Sigma-Aldrich and were of analytical grade.
Liang Q., Cao L., Zhu C., Kong Q., Sun H., Zhang F., Mou H, & Liu Z. (2022). Characterization of Recombinant Antimicrobial Peptide BMGlv2 Heterologously Expressed in Trichoderma reesei. International Journal of Molecular Sciences, 23(18), 10291.
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4

Plasmid Construction and E. coli Cultivation

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E. coli DH5α (Thermo Fisher Scientific) was used for propragation of all plasmids used in the study. The E. coli DH5α strains were routinely cultivated in lysogeny broth (LB) medium (LB Broth, Sigma Aldrich), 37 o C, 180 rpm, and supplemented with appropriate antibiotic(s) (final concentration: spectinomycin 50 μg/mL, carbenicillin 100 μg/mL, chloramphenicol 37 μg/mL, and erythromycin 200 μg/mL). Plasmids were constructed using a modular plasmid assembly method, namely Biopart Assembly Standard Idempotent Cloning (BASIC) as described previously (Storch et al., 2015; (link)Yunus et al., 2020 (link)Yunus et al., , 2018;; Yunus and Jones, 2018) unless stated otherwise. All amino acid sequences of enzymes used in this study are listed in Supplementary Table S1.
Yunus I.S., Anfelt J., Sporre E., Miao R., Hudson E.P, & Jones P.R. (2022). Synthetic metabolic pathways for conversion of CO2 into secreted short-to medium-chain hydrocarbons using cyanobacteria. Metabolic engineering, 72.
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5

Cultivation of Brucella and E. coli Strains

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Wild-type strains derived from B. abortus 544 (ATCC 23448), a smooth, virulent B. abortus biovar 1 strain, were cultivated in either Brucella broth (Becton Dickinson, USA) or on Brucella agar. Bacteria were cultured at 37 o C with vigorous shaking until they reached stationary phase. Escherichia (E.) coli DH5α was purchased from Invitrogen (USA) and used for creating plasmid constructs. E. coli DH5α culture was routinely grown at 37 o C in LB broth or agar supplemented with 100 μg/ml of ampicillin (Sigma, USA).
Arayan L.T., Huy T.X.N., Reyes A.W.B., Hop H.T., Son V.H., Min W., Lee H.J, & Kim S. (2019). Substantial Protective Immunity Conferred by a Combination of Brucella abortus Recombinant Proteins against Brucella abortus 544 Infection in BALB/c Mice. Journal of microbiology and biotechnology, 29(2).
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6

Bacterial Strains and Phage Propagation Protocol

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Escherichia coli BW 25113 and ΔtrxA were obtained from the National BioResource Project (NBRP), Shizuoka, Japan. Escherichia coli DH5α was purchased from TaKaRa Bio (Shiga, Japan), and Bacillus coagulans NBRC 12714 was obtained from National Institute of Technology and Evaluation at the Biological Resource Center (NBRC), Kisarazu, Chiba, Japan. LB medium (Difco) and tryptic soy agar (TSA; Difco) were used for bacterial cultures. All bacterial strains were stored at −80°C in glycerol stocks and then precultured at 37°C overnight in LB medium with shaking from a single colony grown on TSA plates, prior to experimentation. Culture media and agar plates for the recombinant strains were supplemented with 10 μg/ml of kanamycin.
T7 phage was purchased from the NBRC. After overnight infection to exponentially growing E. coli DH5α at 37°C in LB medium with shaking, cells were collected and the supernatant was filtered through a 0.45-μm membrane (Merck Millipore, Ireland) to produce the T7 phage solution. This filtered T7 phage solution (1010 PFU/ml) was stored at 4°C and used for subsequent experiments.
Masuda Y., Kawabata S., Uedoi T., Honjoh K.I, & Miyamoto T. (2021). Construction of Leaderless-Bacteriocin-Producing Bacteriophage Targeting E. coli and Neighboring Gram-Positive Pathogens. Microbiology Spectrum, 9(1), 10.1128/spectrum.00141-21.
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7

Bacterial Strains and Phage Propagation Protocol

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Escherichia coli BW 25113 and ΔtrxA were obtained from the National BioResource Project (NBRP), Shizuoka, Japan. Escherichia coli DH5α was purchased from TaKaRa Bio (Shiga, Japan), and Bacillus coagulans NBRC 12714 was obtained from National Institute of Technology and Evaluation at the Biological Resource Center (NBRC), Kisarazu, Chiba, Japan. LB medium (Difco) and tryptic soy agar (TSA; Difco) were used for bacterial cultures. All bacterial strains were stored at −80°C in glycerol stocks and then precultured at 37°C overnight in LB medium with shaking from a single colony grown on TSA plates, prior to experimentation. Culture media and agar plates for the recombinant strains were supplemented with 10 μg/ml of kanamycin.
T7 phage was purchased from the NBRC. After overnight infection to exponentially growing E. coli DH5α at 37°C in LB medium with shaking, cells were collected and the supernatant was filtered through a 0.45-μm membrane (Merck Millipore, Ireland) to produce the T7 phage solution. This filtered T7 phage solution (1010 PFU/ml) was stored at 4°C and used for subsequent experiments.
Masuda Y., Kawabata S., Uedoi T., Honjoh K.I, & Miyamoto T. (2021). Construction of Leaderless-Bacteriocin-Producing Bacteriophage Targeting E. coli and Neighboring Gram-Positive Pathogens. Microbiology Spectrum, 9(1), 10.1128/spectrum.00141-21.
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8

Characterization of S. aureus NADK

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S. aureus ATCC 12600 and E. coli DH5α were obtained from Merck Biosciences. Pvt Ltd. S. aureus was grown on modified Baird Parker media at 37°C. A clear isolated colony exhibiting distinct zone and shiny black color was picked and inoculated in brain heart infusion (BHI) broth and grown for overnight at 37°C and this grown S. aureus ATCC 12600 culture was used to characterize NADK. E. coli DH5α were used in the expression of S. aureus NADK gene cloned in pQE 30 vector.[18 (link)]
Prasad U.V., Vasu D., Gowtham R.R., Pradeep C.K., Swarupa V., Yeswanth S., Choudhary A, & Sarma P.V. (2017). Cloning, Expression and Characterization of NAD Kinase from Staphylococcus aureus Involved in the Formation of NADP (H): A Key Molecule in the Maintaining of Redox Status and Biofilm Formation. Advanced Biomedical Research, 6, 97.
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