Escherichia coli

Manufactured by Promega

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Escherichia coli is a bacterium commonly used in laboratory settings. It serves as a model organism for various scientific studies and experiments.

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Lab products found in correlation

Pgex 6p 3 constructs, by GE Healthcare (3 mentions) Nllgliea k 13c615n2, by Merck Group (1 mentions) Bj5183 cells, by Addgene (1 mentions) Bglii and hindiii, by Promega (1 mentions) Restriction endonuclease pme 1, by New England Biolabs (1 mentions) Pjet vector, by Thermo Fisher Scientific (1 mentions) Q5 hot start dna polymerase, by New England Biolabs (1 mentions) Dneasy blood tissue kit, by Qiagen (1 mentions) Zymoclean gel dna recovery kit, by Zymo Research (1 mentions)

Close spelling variants of this product

Escherichia coli JM109 JM109 Escherichia coli cells Escherichia coli JM109(DE3) Escherichia coli KRX Escherichia coli S30 System Escherichia coli JM109 competent cells Escherichia coli strain Top10 Escherichia coli BL21 (DE3) Escherichia coli BL21 cells JM109 competent Escherichia coli

6 protocols using escherichia coli

ProGRP Isoform 1 Expression and Purification

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ProGRP isoform 1 (AA 1-125 + 8) was cloned from human cDNA (OriGene Technologies), expressed in Escherichia coli (Promega) using pGEX-6P-3 constructs (GE Healthcare) and purified as described elsewhere.^{16 (link)} The concentration of the ProGRP stock solution was determined by absorbance at 280 nm (A280). Working solutions were prepared by dilution with 50 mM ammonium bicarbonate solution (ABC solution) and stored at 4 °C.
Initially, complex samples were prepared by adding Lys-C or trypsin digested ProGRP to trypsin digested human serum from healthy subjects. The digested standards were added to the digested serum immediately before performing the extraction. In later experiments, spiked serum samples were prepared by adding intact ProGRP immediately before digestion with trypsin beads.

Levernæs M.C., Farhat B., Oulie I., Abdullah S.S., Paus E., Reubsaet L, & Halvorsen T.G. (2019). Immunocapture sample clean-up in determination of low abundant protein biomarkers – a feasibility study of peptide capture by anti-protein antibodies. RSC Advances, 9(60), 34902-34911.

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Recombinant ProGRP Isoform 1 Production

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Recombinant ProGRP isoform 1 (AA 1−125 + 8) was cloned from human cDNA (OriGene Technologies, Rockville, MD,USA), expressed in Escherichia coli (Promega, Madison, WI, USA) using pGEX-6P-3 constructs (GE Healthcare Little Chalfont, UK) and purified as described elsewhere46 (link). Solutions of ProGRP and the Internal Standard (IS) NLLGLIEA[K_¹³C₆¹⁵N₂] (purity >95%, Sigma-Aldrich) were prepared as described elsewhere21 (link).

Rossetti C., Świtnicka-Plak M.A., Grønhaug Halvorsen T., Cormack P.A., Sellergren B, & Reubsaet L. (2017). Automated Protein Biomarker Analysis: on-line extraction of clinical samples by Molecularly Imprinted Polymers. Scientific Reports, 7, 44298.

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Recombinant Ad-GluR6c-GFP Production

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Recombinant Ad-GluR6c-green fluorescent protein constructs were produced in accordance with standard techniques (He et al., 1998). The pAd Track CMV vector is bicistronic, and expresses both green fluorescent protein and the GluR6c domain. Briefly, GluR6c (852-908 amino acids of GluR6) was generated by polymerase chain reaction of the appropriate GluR6c coding region to incorporate lanking Bgl II and Hind III sites followed by ligation into the Ad shuttle vector pAdTrack-CMV digested with Bgl II and Hind III (Promega). The resultant plasmid was linearized by digestion with restriction endonuclease Pme I (New England Biolabs, Beverly, MA), and subsequently cotransformed into Escherichia coli (Promega). BJ5183 cells (Addgene, Cambridge, MA, USA) have an adenoviral backbone plasmid pAdEasy-1. Recombinants were selected with kanamycin, and recombination confirmed by restriction endonuclease analyses. Finally, the linearized recombinant plasmid was transfected into Ad packaging cell lines, Human Embryonic Kidney 293 cells (Addgene). Recombinant Ads were generated typically within 7 to 12 days, purified, and then tittered.

Mou J., Liu X, & Pei D. (2014). Overexpression of C-terminal fragment of glutamate receptor 6 prevents neuronal injury in kainate-induced seizure via disassembly of GluR6-PSD-95-MLK3 signaling module. Neural Regeneration Research, 9(23), 2059-2065.

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Sequencing Mutant Alleles in A. gambiae

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DNA was individually extracted from L3 and L4 A. gambiae larvae using the DNeasy Blood & Tissue Kits (Qiagen, catalog no./ID: 69504). Genomic DNA (1 μl) was used as a template in a 20 μl of PCR reaction using Q5 HotStart DNA polymerase [New England Biolabs (NEB), catalog no./ID: M0493L] and primers 1154A.S5 and 1154A.S7 amplifying genomic fle sequences. The resulting product was run on a 1% agarose gel in tris-acetate-EDTA (TAE) buffer, gel-extracted with the Zymoclean Gel DNA Recovery Kit (Zymo Research, catalog no./ID: D4007), cloned into the pJET vector (Thermo Scientific, catalog no./ID: K1231), transformed into chemically competent Escherichia coli (Promega, JM109), and plated on LB-ampicillin plates. Sanger sequencing reads from individual colonies represented amplicons from individual mutant alleles, and primers PJET1-2F and/or PJET 1-2R were compared to fle sequences from our WT mosquitoes as a reference genome and a selection are summarized in fig. S2. All primer sequences can be found in table S21.

Smidler A.L., Pai J.J., Apte R.A., Sánchez C. H.M., Corder R.M., Jeffrey Gutiérrez E., Thakre N., Antoshechkin I., Marshall J.M, & Akbari O.S. (2023). A confinable female-lethal population suppression system in the malaria vector, Anopheles gambiae. Science Advances, 9(27), eade8903.

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Bacterial Sensing Biosensor Validation

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Escherichia coli (Promega Corp., Madison, WI, USA), Acinetobacter baumannii (ATCC 19606), Staphylococcus aureus (ATCC 6538), and Pseudomonas aeruginosa (ATCC 9027) were utilized for sensing studies. All bacteria were grown in a nutrient broth medium (BD Difco TM , BD Company, Franklin Lakes, NJ, USA). Each culture was serially diluted to obtain 10 7 CFU of each organism. The microdilution method was used to prepare the standard concentration of bacteria. The stock solution of bacteria (10 1 to 10 7 CFU/mL) was prepared in 1 × PBS buffer and used for sensing studies. Each bacterial concentration was loaded to each biosensor electrode and washed with PBS buffer, followed by incubation for 30 min. Subsequently, CV, DPV, and impedance measurements were obtained. To test the clinical utility of the designed biosensor, the biosensor was tested in urine sample analysis. The urine samples of healthy volunteers were collected in a sterile glass container. A range of bacterial concentrations (10 to 10 6 CFU/mL) were added to the urine samples.

Raj P., Choi C.H., Han K., & Lee T.Y. (2021). Label-Free Electrochemical Biosensor Based on Au@MoS₂–PANI for Escherichia coli Detection. Chemosensors, 9(3), 49-49.

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Recombinant ProGRP and hCG Assay

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ProGRP isoform 1 (AA 1-125 + 8) was cloned from the Small Cell Lung Cancer cell line NCI-H128 (ATCC No. HTB-120), expressed in Escherichia coli (Promega) using pGEX-6P-3 constructs (GE Healthcare) and purified as described elsewhere 17 . The concentration of the ProGRP stock solution was determined by absorbance at 280 nm (A280). Working solutions were prepared by dilution with 50 mM ammonium bicarbonate buffer solution (ABC-buffer) and stored at -4 °C.
One syringe of Ovitrelle (250 µg/mL koriongonadotropin alfa) was transferred to a Protein LoBind Eppendorf tube from Eppendorft AG (Hamburg, Germany) and stored at 4 °C. Working solutions of hCG were made by diluting the stock solution with ABC-buffer.
Spiked serum samples were prepared by adding working solutions of ProGRP and hCG to human serum from healthy subjects. The standards were added to serum immediately before the experiments were performed.

Levernæs M.C.S., Broughton M.N., Reubsaet L, & Halvorsen T.G. (2017). To elute or not to elute in immunocapture bottom-up LC-MS. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 1055-1056.

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