HT115(DE3) strains carrying empty vector L4440, ubq-2, or rpt-2 plasmids (from C. elegans of RNAi library generated by M. Vidal et al.) were cultured in LB with 100 μg/ml of Ampicillin for 16–18 hr at 37°C. About 50 μl of bacteria liquid culture was seeded onto a NGM plate with 100 μg/ml of Ampicillin and 1 mM of IPTG (Takara) for induction of dsRNA expression. Stage-synchronized early L1 animals were cultured on RNAi plates at 20°C for 24 hr. The animals were then examined and imaged under OLYMPUS BX63 microscope.
Ampicillin
Manufactured by Takara Bio
Sourced in China
Ampicillin is a beta-lactam antibiotic used in microbiology and molecular biology laboratories. It is a powder that can be dissolved in water or other appropriate solvents. Ampicillin inhibits the synthesis of bacterial cell walls, making it effective against a variety of gram-positive and gram-negative bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Kanamycin, by Takara Bio (4 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Takara Bio (2 mentions)
Bx63 microscope, by Olympus (1 mentions)
Pcold tf, by Takara Bio (1 mentions)
Restriction enzyme, by Thermo Fisher Scientific (1 mentions)
Bl21 de3, by Thermo Fisher Scientific (1 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Avantor (1 mentions)
Diversify pcr random mutagenesis kit, by Takara Bio (1 mentions)
Spectinomycin, by Takara Bio (1 mentions)
Pet 29a, by Takara Bio (1 mentions)
Pmd19 t, by Takara Bio (1 mentions)
Isopropyl β d thiogalactopyranoside iptg, by Takara Bio (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (1 mentions)
Dna polymerase, by Takara Bio (1 mentions)
Dna restriction enzyme, by Takara Bio (1 mentions)
Horseradish peroxidase hrp, by Merck Group (1 mentions)
Mut express 2 fast mutagenesis kit v2, by Vazyme (1 mentions)
Ampicillin, by Maclin Biochemical Technology (1 mentions)
E coli dh5α, by Takara Bio (1 mentions)
C4 hsl, by Merck Group (1 mentions)
7 protocols using ampicillin
1
RNAi Screening in C. elegans
2
E. coli Cloning and Expression Protocol
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Escherichia coli strains DH5α and BL21 (DE3) (Invitrogen Corporation, Carlsbad, CA, USA) were grown at 37 °C in Lennox broth (LB) or on solid medium containing 1.5% agar at 37 °C and used for the cloning and expression of recombinant genes. When necessary, LB medium was supplemented with an appropriate dosage of ampicillin (100 μg/ml) or kanamycin (100 μg/ml).
The expression vector pCold-TF was purchased from Takara Bio, Inc. (Shiga, Japan). Restriction enzymes were purchased from MBI Fermentas, Inc. (Waltham, MA, USA). V. harveyi strains BB170 (sensor1− sensor2+) (ATCC BAA-1117)and BB152 (ATCC BAA-1119)were purchased from the American type culture collection (Manassas, VA, USA) and cultivated in modified autoinducer bioassay (AB) medium (Bassler et al. 1993 (link)). BB170 was used as the AI-2 biosensor strain and BB152 as a positive control for AI-2 production. All chemicals used were of analytical grade and purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA).
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Escherichia coli strains DH5α and BL21 (DE3) (Invitrogen Corporation, Carlsbad, CA, USA) were grown at 37 °C in Lennox broth (LB) or on solid medium containing 1.5% agar at 37 °C and used for the cloning and expression of recombinant genes. When necessary, LB medium was supplemented with an appropriate dosage of ampicillin (100 μg/ml) or kanamycin (100 μg/ml).
The expression vector pCold-TF was purchased from Takara Bio, Inc. (Shiga, Japan). Restriction enzymes were purchased from MBI Fermentas, Inc. (Waltham, MA, USA). V. harveyi strains BB170 (sensor1− sensor2+) (ATCC BAA-1117)and BB152 (ATCC BAA-1119)were purchased from the American type culture collection (Manassas, VA, USA) and cultivated in modified autoinducer bioassay (AB) medium (Bassler et al. 1993 (link)). BB170 was used as the AI-2 biosensor strain and BB152 as a positive control for AI-2 production. All chemicals used were of analytical grade and purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA).
3
Plasmid Extraction and Mutagenesis
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A gel recovery kit, a plasmid extraction kit, and a PCR product recovery kit (all from Omega), a protein purification kit (Novagen, Germany), and a Diversify® PCR random mutagenesis kit (Clontech). A 170 kD prestained protein ladder was purchased from Guangzhou Saizhe Biotechnology Co., Ltd. Other reagents included ABTS (Amresco), IPTG and ampicillin (both TaKaRa). Other conventional reagents were all analytically pure.
4
Bacterial Strains and Plasmids for Molecular Cloning
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The bacterial strains and plasmids used in this study are listed in Table 3 . The E. coli strains were grown at 37 °C in LB broth or on LB agar. Other bacterial strains were grown aerobically at 30 °C in LB broth or on LB agar. The following antibiotics were used at the indicated concentrations: ampicillin (Ap), 100 μg/mL; spectinomycin (Sm), 100 μg/mL; gentamicin (Gm), 20 μg/mL; and kanamycin (Km), 50 μg/mL.
All of the enzymes used in the DNA manipulations were obtained from TaKaRa Biotechnology Co. Ltd (Dalian, China). E. coli DH5α and BL21 (DE3) were purchased from TaKaRa and were used for cloning and expression hosts, accordingly. The plasmids pMD19-T (TaKaRa) and pET-29a (+) (TaKaRa) were used as cloning and expression vectors, respectively.
All of the enzymes used in the DNA manipulations were obtained from TaKaRa Biotechnology Co. Ltd (Dalian, China). E. coli DH5α and BL21 (DE3) were purchased from TaKaRa and were used for cloning and expression hosts, accordingly. The plasmids pMD19-T (TaKaRa) and pET-29a (+) (TaKaRa) were used as cloning and expression vectors, respectively.
5
Rapid Quantification of Dialkyl Phosphates
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Analytical DOP standards were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). The hybridoma cell line (12C2), the coating hapten H1 (4-((diethoxyphosphorothioyl) amino) butanoic acid), and the coating antigen (hapten 2-ovalbumin) were self-prepared [11 (link)], as previously described. The XL1-Blue and Top 10F’ Escherichia coli strains have previously been established by our laboratory. The plasmid vector pComb3XSS (Figure 1 A) was obtained from the Barbas Laboratory, TSRI, La Jolla, CA, USA. The helper phage VCSM13 was obtained from the Naval General Hospital of Beijing, China. Horseradish peroxidase (HRP), and 3,3′,5,5′-tetramethylbenzidine (TMB) were obtained from Sigma-Aldrich (Shanghai, China). Ampicillin, kanamycin, and Isopropyl β-d -thiogalactopyranoside (IPTG) were purchased from Takara (Dalian, China). DNA polymerase and DNA restriction enzyme were also purchased from Takara. HRP-conjugated goat anti-mouse IgG and anti-His tag mouse monoclonal antibodies were purchased from TransGen Biotech Co. Ltd (Beijing, China). Mut Express II Fast Mutagenesis Kit V2 for site-specific mutagenesis was obtained from Vazyme Biotech Co., Ltd. (Nanjing, China). All other chemicals were standard commercial analytical-grade reagents.
6
Recombinant E. coli DH5α Transformation
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E. coli DH5α and plasmid pUC118 containing ampR (conferring resistance to ampicillin) were purchased from Takara Bio Inc. (Dalian, China). The recombinant E. coli DH5α containing pUC118 was obtained by transformation experiment as described in the “Transformation experiments under MIBK exposure” section. Mueller Hinton (MH) agar medium and super optimal broth with catabolite repression (SOC) medium were obtained from Oxide (Basingstoke, UK) and Takara Bio Inc. (Dalian, China), respectively. Luria-Bertani (LB) liquid medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) was prepared according to the guidelines of Molecular Cloning (Green and Sambrook 2012 ). ampicillin, MIBK, and other chemicals of reagent-grade were supplied by Macklin Inc. (Shanghai, China).
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E. coli DH5α and plasmid pUC118 containing ampR (conferring resistance to ampicillin) were purchased from Takara Bio Inc. (Dalian, China). The recombinant E. coli DH5α containing pUC118 was obtained by transformation experiment as described in the “Transformation experiments under MIBK exposure” section. Mueller Hinton (MH) agar medium and super optimal broth with catabolite repression (SOC) medium were obtained from Oxide (Basingstoke, UK) and Takara Bio Inc. (Dalian, China), respectively. Luria-Bertani (LB) liquid medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) was prepared according to the guidelines of Molecular Cloning (Green and Sambrook 2012 ). ampicillin, MIBK, and other chemicals of reagent-grade were supplied by Macklin Inc. (Shanghai, China).
7
Recombinant Protein Expression and Purification
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The TIANprep Midi Plasmid Kit was used for plasmid or genomic DNA extractions and the E com BL21 (DE3) Chemically Competent Cell was the host-expressing cell; the Uniclone One Step Seamless Cloning Kit recombinant plasmid construction kit and the total RNA Extraction Kit were purchased from Genesand company. T4 DNA Ligase, BamH I restriction endonuclease, and other reagents were used to construct the expression vectors. Kanamycin (30 mg/mL), ampicillin (10 mg/mL), 5-Bromo-4-chloro-3-indolyl β-Dgalactoside (X-Gal), and Isopropyl β-D-Thiogalactoside (IPTG) solutions were purchased from TaKaRa and used as the recombinant strain selection medium. The Mag-Beads His-Tag protein puri cation beads were used for the puri cation of the His-Tag target protein, and the target protein concentration was determined by BCA Protein Assay Kit. Western blot analysis was performed on the target protein (PvdQ enzyme). Anti-6 His-Tag mouse monoclonal antibody was used as the primary antibody, HRP-conjugated rabbit anti-mouse IgG was the second antibody, and the EasyBlot ECL kit was used to observe the color reaction. The AHL standard reagents were purchased from Sigma (C4-HSL, C6-HSL, C8-HSL, 3-HSL, 3oxo-C8-HSL, C10-HSL, 3-oxo-C10-HSL, C12-HSL, C14-HSL, 3-oxo-C14-HSL) and dissolved with methanol (HPLC grade, 99.9%), sealed, and stored at -20°C.
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