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Gene pulser xcell electroporator

Manufactured by Bio-Rad
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The Gene Pulser Xcell electroporator is a lab equipment designed for electroporation, a process of introducing foreign material into cells by creating temporary pores in the cell membrane using electrical pulses. The device generates and delivers the required electrical pulses to facilitate this process.

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Lab products found in correlation

Gene pulser micropulser cuvette, by Bio-Rad (5 mentions) Opti mem, by Thermo Fisher Scientific (5 mentions) Megascript t7 transcription kit, by Thermo Fisher Scientific (4 mentions) Electroporation buffer, by Teknova (3 mentions) Renilla luciferase assay system, by Promega (3 mentions) Fastdigest restriction enzyme, by Thermo Fisher Scientific (3 mentions) Phusion dna polymerase, by Thermo Fisher Scientific (3 mentions) Rapid dna ligation kit, by Thermo Fisher Scientific (3 mentions) Firefly luciferase mrna, by TriLink (2 mentions) Luciferase assay system, by Promega (2 mentions) Genejet plasmid miniprep kit, by Thermo Fisher Scientific (2 mentions) Bioanalyzer 2100 rna 6000 nano kit, by Agilent Technologies (2 mentions) Rneasy minelute cleanup kit, by Qiagen (2 mentions) Quikchange lightning site directed mutagenesis kit, by Agilent Technologies (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions) Exo fect exosome transfection kit, by System Biosciences (2 mentions) 3t3 l1 fibroblast, by American Type Culture Collection (1 mentions) Corresponding restriction enzymes, by Thermo Fisher Scientific (1 mentions) Monarch dna gel extraction kit, by New England Biolabs (1 mentions)

66 protocols using gene pulser xcell electroporator

1

Yeast Electroporation and Transformation

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Electrocompetent cells were prepared from a saturated culture of S. cerevisiae grown in YEP medium at 30°C at an absorbance of 1.3–1.5 at 600 nm. Cells were collected by centrifugation and washed twice with a one-half volume of sterile cold water. Subsequently, the cells were washed with a one-quarter volume of cold 1 M sorbitol, suspended in 1–2 ml of 1 M sorbitol, and separated into 100 μl fractions. For the DNA assembler method, an aliquot of electrocompetent cells was mixed with 2 μl of each PCR fragment (previously dialyzed) to be assembled and transferred to a 2 mm electroporation cuvette. A pulse of 1,500 V, 25 μF, and 200 Ω was applied using a Gene Pulser Xcell electroporator (BioRad Laboratories, Inc, Hercules, CA). X. dendrorhous transformation was also performed by electroporation as previously described (31 , 32 (link)) using 1–5 μg of linear transformant DNA. Electrocompetent cells were prepared from exponential cultures, grown in YM medium, and electroporated using a Gene Pulser Xcell electroporator with Pulse Controller and Capacitance Extender modules (BioRad Laboratories, Inc, Hercules, CA) under the following conditions: 125 mF, 600 Ω, and 0.45 kV.
González A.M., Venegas M., Barahona S., Gómez M., Gutiérrez M.S., Sepúlveda D., Baeza M., Cifuentes V, & Alcaíno J. (2022). Damage response protein 1 (Dap1) functions in the synthesis of carotenoids and sterols in Xanthophyllomyces dendrorhous. Journal of Lipid Research, 63(3), 100175.
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2

Standard Molecular Biology Techniques

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Routine DNA manipulations were performed using standard procedures unless otherwise indicated. Oligonucleotides were synthesized by StabVida (Caparica, Portugal). FastDigest restriction enzymes, Phusion DNA polymerase, and the Rapid DNA Ligation Kit (Thermo Scientific) were used according to the manufacturer’s instructions. Plasmids were purified using a Macherey-Nagel Plasmid Purification Kit according to the manufacturer’s protocol. Plasmids were transformed in E. coli by electroporation (1 mm cuvette; 200 Ω, 25 µF, 1,250 V; Gene Pulser X-Cell electroporator, BioRad). S. aureus competent cells were generated as previously described (37 (link)). Plasmids were transformed in S. aureus by electroporation (1 mm cuvette; 100 Ω, 25 µF, 1,250 V; Gene Pulser X-Cell electroporator, BioRad). All constructed plasmids were confirmed by Sanger sequencing at StabVida (Caparica, Portugal).
Gómez-Arrebola C., Hernandez S.B., Culp E.J., Wright G.D., Solano C., Cava F, & Lasa I. (2023). Staphylococcus aureus susceptibility to complestatin and corbomycin depends on the VraSR two-component system. Microbiology Spectrum, 11(5), e00370-23.
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3

Standard Molecular Biology Techniques

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Routine DNA manipulations were performed using standard procedures unless otherwise indicated. Oligonucleotides were synthesized by StabVida (Caparica, Portugal). FastDigest restriction enzymes, Phusion DNA polymerase, and the Rapid DNA Ligation Kit (Thermo Scientific) were used according to the manufacturer’s instructions. Plasmids were purified using a Macherey-Nagel Plasmid Purification Kit according to the manufacturer’s protocol. Plasmids were transformed in E. coli by electroporation (1 mm cuvette; 200 Ω, 25 µF, 1,250 V; Gene Pulser X-Cell electroporator, BioRad). S. aureus competent cells were generated as previously described (37 (link)). Plasmids were transformed in S. aureus by electroporation (1 mm cuvette; 100 Ω, 25 µF, 1,250 V; Gene Pulser X-Cell electroporator, BioRad). All constructed plasmids were confirmed by Sanger sequencing at StabVida (Caparica, Portugal).
Gómez-Arrebola C., Hernandez S.B., Culp E.J., Wright G.D., Solano C., Cava F, & Lasa I. (2023). Staphylococcus aureus susceptibility to complestatin and corbomycin depends on the VraSR two-component system. Microbiology Spectrum, 11(5), e00370-23.
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4

Efficient CRISPR/Cas9 Transfection of Tachyzoites

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For each transfection, 10-20 million purified tachyzoites in 300 μL cytomix buffer (Shen et al., 2017 (link)) were mixed with 7.5 μg purified CRISPR/Cas9 plasmid and 10 μg donor DNA fragment in a 2 mm gap gene pulser® cuvette (Bio-Rad, USA) and electroporated with the Gene Pulser Xcell electroporator (Bio-Rad). After electroporation, the parasites were immediately transferred to confluent HFF host cells. Transfected parasites were grown in HFF cultures for 24 h for recovery prior to drug selection for stable transformants.
Yang Y., Lin M., Chen X., Zhao X., Chen L., Zhao M., Yao C., Sheng K., Yang Y., Ma G, & Du A. (2022). The first apicoplast tRNA thiouridylase plays a vital role in the growth of Toxoplasma gondii. Frontiers in Cellular and Infection Microbiology, 12, 947039.
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5

Differentiation and Electroporation of 3T3-L1 Adipocytes

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3T3-L1 fibroblasts (#CL-173, from American Type Culture Collection, Manassas, VA) were cultured and differentiated as described previously [20 (link)]. Briefly, cells were grown in the growth media [Dulbecco’s modified Eagle medium (DMEM) supplemented with 4.5 g/L glucose, 10% fetal bovine serum, 1% glutamine, and 0.5% penicillin/streptomycin]. Differentiation was induced in post-confluent cells with growth media containing 500 μM isobutylmethylxanthine, 0.2 μM dexamethasone, and 2.5 μg/ml insulin for 3–4 days, and cells were replenished with growth media every 3–4 days. Experiments were performed in adipocytes 12–16 days post differentiation.
Electroporation of 3T3-L1 adipocytes was performed with cells on 10–12 days post differentiation. Differentiated 3T3-L1 adipocytes were electroporated at 200 V and 950 μF with 2 nmole siRNA using a Gene Pulser Xcell electroporator (Bio-Rad, Hercules, CA) and plated onto 12- or 24- well plates for experiments. The siRNA sequences were as follows: MEK1 and 2 (MEK1/2), 5’-AGU CGG ACA UCU GGA GCA U-3’, or 5’-CAG UCG GAC AUC UGG AGC A-3’; IKKβ, 5’-CGA CAG GAG CUC AGC CCA A-3’, or 5’-GGA CAU CGU UGU UAG UGA A-3’; caveolin-1, 5’-AAC CAG AAG GGA CAC ACA G-3’ [21 (link)].
Lu J.C., Chiang Y.T., Lin Y.C., Chang Y.T., Lu C.Y., Chen T.Y, & Yeh C.S. (2016). Disruption of Lipid Raft Function Increases Expression and Secretion of Monocyte Chemoattractant Protein-1 in 3T3-L1 Adipocytes. PLoS ONE, 11(12), e0169005.
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6

RV-B14 Replicon Transfection and Luciferase Assay

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RV-B14 replicon (pΔP1Luc/VP3)42 (link) was a generous gift from Dr. Frank van Kuppeveld. RV-B14 replicon RNA was first linearized by SmaI (New England Biolabs) restriction digestion and then precipitated by 0.1 volume 3M sodium acetate, 0.05 volume 0.5M EDTA, and 2.5 volume 100% ethanol. Next, the linearized plasmid was in vitro transcribed using MEGAscript™ T7 Transcription Kit (Thermo Fisher). Cells were washed twice with 1×phosphate buffered saline (HyClone) and re-suspended in electroporation buffer (Teknova E0399). 3 μg of the replicon RNA was were electroporated into 2 million cells using square wave protocol (Bio-Rad Gene Pulser Xcell electroporator). Electroporated cells were resuspended in H1-HeLa culture medium without antibiotics and seeded into 6-well plates. At 24 hours post-electroporation, total cell lysates were harvested in passive lysis buffer and luciferase expression was measured using Luciferase Assay System (Promega E1500). Luciferase activity was measured by addition of substrate and readings were taken immediately using Glomax 20/20 luminometer using a 5 second integration time. In parallel, 3 μg Firefly luciferase mRNA (capped, Trilink) was electroporated into the same set of cells as a control to test if the loss of SETD3 could have universal impact on cap-dependent translations.
Diep J., Ooi Y.S., Wilkinson A.W., Peters C.E., Foy E., Johnson J.R., Zengel J., Ding S., Weng K.F., Laufman O., Jang G., Xu J., Young T., Verschueren E., Kobluk K.J., Elias J.E., Sarnow P., Greenberg H.B., Hüttenhain R., Nagamine C.M., Andino R., Krogan N.J., Gozani O, & Carette J.E. (2019). Enterovirus Pathogenesis Requires the Host Methyltransferase SETD3. Nature microbiology, 4(12), 2523-2537.
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7

Recombinant DNA Manipulation in P. aeruginosa

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Recombinant DNA manipulations were performed using standard procedures [17 ] and following the manufacturers’ instructions. DNA fragments were amplified by PCR using Phusion™ High-Fidelity DNA Polymerase (Thermo Scientific, Madrid, Spain) for cloning and DreamTaq Green PCR Master Mix (Thermo Scientific, Madrid, Spain) for colony PCR. All primers used in this study are listed in Table S2. Isolation of DNA fragments from agarose gels was carried out using the Monarch® DNA Gel Extraction Kit (New England BioLabs, Ipswich, MA, USA). DNA fragments were digested with corresponding restriction enzymes (Thermo Scientific, Madrid, Spain) and ligated with T4 DNA ligase (Invitrogen, Carlsbad, CA, USA). Plasmid DNA was isolated using the GeneJET™ Plasmid Miniprep Kit (Thermo Scientific, Madrid, Spain) and quantified using a NanoDrop™ 1000 spectrophotometer (Thermo Scientific, Madrid, Spain). DNA was transformed into P. aeruginosa PAO1 cells by electroporation using a Gene Pulser Xcell™ electroporator (Bio-Rad, Hercules, CA, USA) as previously described [18 (link)].
Moya-Andérico L., Admella J., Fernandes R, & Torrents E. (2020). Monitoring Gene Expression during a Galleria mellonella Bacterial Infection. Microorganisms, 8(11), 1798.
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8

Genetic Manipulation Techniques in E. coli and Mycobacteria

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Restriction enzymes, Taq polymerase and T4 DNA ligase were purchased from New England Biolabs (NEB). Standard protocols were followed for DNA manipulation, including plasmid DNA preparation, restriction endonuclease digestion, agarose gel electrophoresis, isolation and ligation of DNA fragments, and E. coli transformation. DNA fragments used for cloning reactions were purified by using the Nucleospin gel extraction kit (Macherey-Nagel) according to the manufacturer’s specifications. Mycobacterial strains were transformed by electroporation at 1800 V, 1000 µF, and 25 Ω on a Bio-Rad Gene pulser X-cell electroporator.
Kishore V., Gaiwala Sharma S.S, & Raghunand T.R. (2023). Septum site placement in Mycobacteria – identification and characterisation of mycobacterial homologues of Escherichia coli MinD. Microbiology, 169(8), 001359.
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9

Generation of CV-B3 Reporter Virus

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The full-length CV-B3-RLuc infectious clone, pRLuc-53CB3/T7 (see Viruses, reporter viruses, and infectious clones), was used a template to introduce indicated triple-alanine substitutions in 2A using QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent) or other standard cloning techniques. Engineered infectious cDNAs were then linearized with SmaI, purified, and transcribed using MEGAscript T7 Transcription Kit (Thermo Fisher). RNA was purified using RNeasy MinElute Cleanup Kit (Qiagen), and RNA quantity and quality was assessed using a Bioanalyzer 2100 RNA 6000 Nano Kit (Agilent). H1-HeLa WT cells (2×106) in electroporation buffer (Teknova) were electroporated with 2µg of the indicated purified viral RNAs using the square wave protocol (120V, 1.5ms pulse, 10 pulse, 1.5s interval, 1mm) on a Gene Pulser Xcell electroporator (Bio-Rad). Firefly luciferase mRNA was included as an electroporation control. Cells were plated in 96-well plates (5×104 cells/well) and were incubated for indicated time points. Cell lysates were harvested and luciferase signals were measured using the Renilla Luciferase Assay System (Promega) and a Veritas Microplate Luminometer (Turner).
Diep J., Ooi Y.S., Wilkinson A.W., Peters C.E., Foy E., Johnson J.R., Zengel J., Ding S., Weng K.F., Laufman O., Jang G., Xu J., Young T., Verschueren E., Kobluk K.J., Elias J.E., Sarnow P., Greenberg H.B., Hüttenhain R., Nagamine C.M., Andino R., Krogan N.J., Gozani O, & Carette J.E. (2019). Enterovirus Pathogenesis Requires the Host Methyltransferase SETD3. Nature microbiology, 4(12), 2523-2537.
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10

Electroporation and DNase I Treatment of Intestinal EVs

Cited 2 times
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The intestinal EV pellet was dissolved in 100 μL PBS. As previously described 34 (link)-36 (link), these EVs were loaded into a Gene Pulser/micropulser Cuvettes (Bio-Rad) for electroporation (GenePulser Xcell electroporator, Bio-Rad) and then treated with DNase I (300U) for 30 mins, 37°C.
Luo Z., Ji Y., Gao H., Reis F.C., Bandyopadhyay G., Jin Z., Ly C., Chang Y.J., Zhang D., Kumar D, & Ying W. (2020). CRIg+ macrophages prevent gut microbial DNA-containing extracellular vesicle-induced tissue inflammation and insulin resistance. Gastroenterology, 160(3), 863-874.
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