Fluorescent M. fortuitum was generated using the pVV16-eGFP replicative vector (Vilchèze et al., 2014 (link)). Electro-competent M. fortuitum were generated as previously described (Viljoen et al., 2018 (link)). M. fortuitum was placed on ice for 2 h prior to pelleting by centrifugation (3,000 × g at 4°C for 15 min). Following centrifugation, bacteria were resuspended in decreasing volumes of wash buffer (10% glycerol (v/v) and 0.025% Tyloxapol in distilled water) and pelleted by centrifugation for a total of 4 washes. For electroporation transformation, 1 μg of plasmid DNA was added to 200 μL of electrocompetent bacteria and transferred to a chilled 0.2 cm electrode gap GenePulser electroporation cuvette (Bio-rad) and transformed using a GenePulser Cxell electroporator (Bio-rad) (2.5 kV, 1, 000 Ω and 25 μF). Bacteria were recovered in 800 μL of 7H9OADC/T and placed at 37°C overnight. For selection of green fluorescent colonies, M. fortuitum was plated on 7H10OADC supplemented 50 μg/mL kanamycin (Euromedex). Positive colonies were selected based on fluorescence and maintained in 7H9OADC/T supplemented with 50 μg/mL kanamycin. Fluorescent M. abscessus has been previously described (Bernut et al., 2014a (link)).
Genepulser electroporation cuvette
Manufactured by Bio-Rad
The GenePulser electroporation cuvette is a laboratory equipment designed for the purpose of delivering an electric pulse to cells, facilitating the introduction of foreign genetic material into the cells. The cuvette provides a contained environment for the electroporation process.
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5 protocols using genepulser electroporation cuvette
1
Generating Fluorescent Mycobacterium Mutants
2
Electroporation of 3T3-L1 Adipocytes
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3T3-L1 murine adipocytes were purchased from ATCC (via LGC Standards, U.S.A. RRID:CVCL_0123) and grown and differentiated as outlined [34 (link),41 (link)]. Stable lines of 3T3-L1 fibroblasts expressing HA-GLUT4-GFP had previously been generated in the lab [42 (link)]. Cells were incubated in a 10% CO2 humid atmosphere incubator at 37°C.
For electroporation, adipocytes were used at day 6 post-differentiation. Cells were washed in PBS before detaching using 0.05% (w/v) trypsin: 2 mg/ml collagenase. Once detached, cells were washed and transferred to 0.2 cm BioRad Gene Pulser® electroporation cuvette containing 3 nmol Silencer® select pre-designed siRNA and electroporated using settings of 0.18 kV and 975 μF. Cells were then plated and assayed 72 h after electroporation [43 (link)]. siRNA against EFR3 was siRNA ID s94606 (ThermoFisher) and PI4K-IIIα siRNA ID s104706 (ThermoFisher).
2-Deoxy-D -glucose (2DG) uptake was assayed as in [34 (link)]. Non-specific association of radioactivity with the cells were quantified by performing parallel assays in the presence of 10 μM cytochalasin B [44 (link)].
For electroporation, adipocytes were used at day 6 post-differentiation. Cells were washed in PBS before detaching using 0.05% (w/v) trypsin: 2 mg/ml collagenase. Once detached, cells were washed and transferred to 0.2 cm BioRad Gene Pulser® electroporation cuvette containing 3 nmol Silencer® select pre-designed siRNA and electroporated using settings of 0.18 kV and 975 μF. Cells were then plated and assayed 72 h after electroporation [43 (link)]. siRNA against EFR3 was siRNA ID s94606 (ThermoFisher) and PI4K-IIIα siRNA ID s104706 (ThermoFisher).
2-Deoxy-
3
Competent ΔespG1/G2 Bacterial Preparation
4
Electroporation of V. cholerae with Plasmids
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The V. cholerae strains were transformed by electroporation as described in [25 (link)] with modification. Briefly, V. cholerae cells from 2 mL overnight cultures were pelleted by centrifugation, resuspended, and then pelleted three times in 1 mL of a cold solution of 2 mM CaCl2 on ice. The supernatant was removed, and the bacterial pellet was resuspended in 40 μL of 2 mM CaCl2 on ice. Then, 1 μg of plasmid DNA was added in a volume of 1 μL to the bacterial suspension, mixed, and transferred to a 0.2 cm chilled Gene Pulser electroporation cuvette (Bio-Rad). Electroporation was performed on a Gene Pulser Xcell device (Bio-Rad). The pulse settings were as follows: pulse type: exponential, C (μF) = 25, PC (ohm) = 200, and V = 2500. Immediately after electroporation, the mixtures were suspended in 2 mL of LB broth incubated at 37 °C for 30 min with shaking at 200 rpm, plated on Petri dishes with appropriate media, and grown at RT for 12–24 h.
5
Plasmid Interference Assay Protocol
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Plasmid interference assays were carried out as previously described (46) . Chemically competent BL21-AI E. coli were transformed with pCsm variants and transformants were selected on Miller's LB broth (Invitrogen) agar supplemented with 34 μg/ml chloramphenicol.
Single colonies were cultured in super optimal broth medium (SOB) (BD Difco) and made electrocompetent through successive washes with 10% glycerol. In triplicate, 100 ng of pTrcHis plasmid (with or without transcribed complementary target sequences) were added to 50 μl of competent cells in a 0.2 cm-gap Gene Pulser® electroporation cuvette (BioRad) on ice.
Cuvettes were transferred to a Gene Pulser II (BioRad) and pulsed with the following settings: 25 μF capacitance, 2.5 kV, and 200 ohms. Immediately following transformation, 950 μl of super optimal broth with catabolite repression (SOC) (SOB + 20 mM glucose) was used to recover pulsed cells from the cuvette and moved to 1.5 ml microcentrifuge tubes. The tubes were shaken at 200 rpm at 37 C for 60 minutes. Serial 10-fold dilutions were made to 10-5 and spot plated onto LB agar containing 100 μg/mL ampicillin and 34 μg/mL chloramphenicol to select for pTrcHis and pCsm, respectively. Plates were imaged after overnight incubation at 37 C.
Single colonies were cultured in super optimal broth medium (SOB) (BD Difco) and made electrocompetent through successive washes with 10% glycerol. In triplicate, 100 ng of pTrcHis plasmid (with or without transcribed complementary target sequences) were added to 50 μl of competent cells in a 0.2 cm-gap Gene Pulser® electroporation cuvette (BioRad) on ice.
Cuvettes were transferred to a Gene Pulser II (BioRad) and pulsed with the following settings: 25 μF capacitance, 2.5 kV, and 200 ohms. Immediately following transformation, 950 μl of super optimal broth with catabolite repression (SOC) (SOB + 20 mM glucose) was used to recover pulsed cells from the cuvette and moved to 1.5 ml microcentrifuge tubes. The tubes were shaken at 200 rpm at 37 C for 60 minutes. Serial 10-fold dilutions were made to 10-5 and spot plated onto LB agar containing 100 μg/mL ampicillin and 34 μg/mL chloramphenicol to select for pTrcHis and pCsm, respectively. Plates were imaged after overnight incubation at 37 C.
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