Due to the difficulty of transfecting DNA or RNA into HUVECs, electroporation was used for introducing siRNA into these cells. The siRNAs transfected were Human TRPM7 (Dharmacon, L-005393-00) and Non-targeting pool (Dharmacon, D-001810-10-05). As in the case of infection by adenovirus, the cells were passaged from a 80 to 90% confluent dish into 100x20 or 60x15 mm cell culture dishes between 16 and 18 hours before the electroporation. At the electroporation time, approximately 120,000 cells were resuspended in Gene Pulser electroporation buffer (Bio-rad, 165–2676) and placed in a Gene Pulser cuvette, 0.2 cm electrode gap (Bio-rad, 165–2082), along with siRNA at 100 nM or 200 nM. The electroporation of HUVECs was then conducted in a Gene Pulser Xcell Total System (Bio-Rad, 165–2660), with a single pulse of a square wave at 150 V for 20 ms. The electroporated cells were plated on a 35x10 mm cell culture dish (Corning, 430165). Experiments were conducted 2 days after electroporation.
Gene pulser cuvette
Manufactured by Bio-Rad
Sourced in United States, Germany
The Gene Pulser cuvette is a specialized laboratory equipment used in electroporation, a technique that facilitates the introduction of genetic material, such as DNA or RNA, into cells. The cuvette provides a container for the sample and is designed to optimize the electrical field for effective cell membrane permeabilization during the electroporation process.
Automatically generated - may contain errors
Lab products found in correlation
Gene pulser xcell, by Bio-Rad (6 mentions)
Micropulser electroporator, by Bio-Rad (5 mentions)
Gene pulser, by Bio-Rad (5 mentions)
Micropulser, by Bio-Rad (4 mentions)
Opti mem, by Thermo Fisher Scientific (3 mentions)
Eporator, by Eppendorf (3 mentions)
Gene pulser xcell electroporation system, by Bio-Rad (3 mentions)
Albumax 2, by Thermo Fisher Scientific (2 mentions)
Amaxa nucleofector device, by Lonza (2 mentions)
Kapa express extract kit, by Roche (2 mentions)
Hygromycin b, by Cayman Chemical (2 mentions)
Geneticin g418, by GoldBio (2 mentions)
Zeocin, by Cayman Chemical (2 mentions)
Nourseothricin, by GoldBio (2 mentions)
Non targeting pool, by Horizon Discovery (1 mentions)
Gene pulser xcell total system, by Bio-Rad (1 mentions)
Gene pulser electroporation buffer, by Bio-Rad (1 mentions)
Dna clean concentrator 5 kit, by Zymo Research (1 mentions)
Gene pulser xcell device, by Bio-Rad (1 mentions)
Nucleofector 2, by Lonza (1 mentions)
65 protocols using gene pulser cuvette
1
TRPM7 Knockdown in HUVECs by Electroporation
2
Rapid Genome Editing in E. coli
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The editing process was performed as previously described [70 (link)]. In short, MG1655 competent cells harbouring pCas9 were prepared, and arabinose (10 mM final concentration) was added to the culture for λ-Red induction. For electroporation, 50 μL of cells was mixed with 200 ng of ptargetET-tdh-ilvA. Electroporation was performed in a 2-mm Gene Pulser cuvette (Bio-Rad) at 2.5 kV and the product was suspended immediately in 1 mL of LB medium. Cells were recovered at 30 °C for 1 h before being spread onto LB agar containing kanamycin (50 mg/L) and spectinomycin (50 mg/L) and incubated overnight at 30 °C. Transformants were identified by colony PCR and DNA sequencing with primer pairs ilvA-I-F/R and tdh-I-F/R for ilvA and tdh, respectively. For the curing of ptargetET-tdh-ilvA, the edited colony was inoculated into 2 mL of LB medium containing kanamycin (50 mg/L) and IPTG (isopropyl-D-thiogalactopyranoside; 0.5 mmol/L). The culture was incubated for 8 to 16 h, diluted and spread onto LB plates containing kanamycin (50 mg/L). The colonies were confirmed as cured by determining their sensitivity to spectinomycin (50 mg/L). pCas was cured by non-selectively growing the colonies overnight at 37 °C.
3
Transfection of Cells with LifeAct-TagGFP2
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Cultured cells were harvested and resuspended in RPMI-1640 medium and plasmid DNA pCMV LifeAct-TagGFP2 was added to a final concentration of 80 μg/ml. Then cells were kept on ice for 5 minutes, followed by delivery to a Gene Pulser Cuvette (Bio-Rad no 165–2088 with 0.4 cm electrode) and electrical pulses were applied to the cells using a MicroPulser™ Electroporator (BIO-RAD) with the following settings: resistance- ∞, capacitance—950 μF, voltage—250 V. After electroporation, cells were immediately transferred into 5 ml of RPMI-1640 with 5% FCS and antibiotics and then replaced with fresh medium after 24 hours. The entire procedure was carried out at room temperature.
4
Bacterial Transformation via Electroporation
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Before electroporation, 20 μl of the DNA solution was placed on a Millipore VSWP02500 membrane (25 mm diameter, 0.025 μm porosity) floating on a Petri dish filled with 40 ml of ultrapure sterile water. After 1 hour of dialysis, the droplet is recovered by pipetting. 1 μl of dialyzed DNA was mixed with 50μl electro-competent E. coli Top 10 cells in pre-chilled 1 mm electrodes distance Gene Pulser cuvette (Biorad) and the electroporation was then performed with Gene Pulser Xcell device (voltage 1800V, capacitance 25μF, resistance 200 ohm). Electroporated cells were recovered with 1ml of LB and incubated at 37 C with orbital shaking during 45 minutes before plating on LB plates containing the appropriate antibiotics. The number of transformants is estimated by plating serial 10-fold dilutions and counting colony forming units (cfu) after overnight incubation at 37 C. Typically, between 106 and 107 cfu were obtained per electroporation. Alternatively, the DNA was concentrated at least 10-fold using the “DNA Clean & Concentrator™-5” kit from Zymo Research and up to 108 cfu can be obtained with 5 μl in a single electroporation.
5
Gene Disruption in Primary CD4+ T Cells
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Gene disruption in primary CD4+ T cells was performed by transfection with in vitro-prepared Cas9 ribonucleoprotein (RNP) complexes. For all targets, gene-specific Alt-RCRISPR-Cas9 gRNA was obtained from IDT (sequences given in Supplementary Table 2 ). To generate RNP complexes, 150 pmol Alt-RCRISPR-Cas9 gRNA was incubated with 150 pmol Alt-R tracrRNA (IDT) in nuclease-free duplex buffer (IDT) at 95°C for 5 min and resultant duplex allowed to cool to room temperature. One hundred and fifty pmol of Alt-R S.p Cas9 Nuclease V3 (IDT) and duplexed gRNA were mixed in 8 μl nuclease-free duplex buffer and incubated at 37°C for 15 min. One hundred and fifty pmol Alt-RCas9 Electroporation Enhancer (IDT) was added to the RNP solution, and the whole mix then added to 1.5 × 106 naïve primary CD4+ T cells, which had previously been washed with room-temperature OptiMEM three times and resuspended in 50 μl OptiMEM. The cell-RNP mix was transferred to a Gene Pulser cuvette (BioRad) and pulsed for 2 ms at 300 V in an ECM 830 Square Wave Electroporation System (BTX). Cells were then immediately transferred to 500 μl supplemented RPMI-1640. Hundred μl of cells were removed and blasted for 3 days as described above, while the remaining ~1.2 × 106 cells were left in resting culture until used in T cell stimulation assays.
6
Transfection of P. falciparum Dd2 Parasites
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P. falciparum Dd2 was cultured at 2 to 5% hematocrit in O+ erythrocytes in Malaria Culture Medium: RPMI 1640 supplemented with 5 g/L Albumax II (Gibco), 0.12 mM hypoxanthine (1.2 ml 0.1 M hypoxanthine in 1 M NaOH), and 10 μg/ml gentamicin (60 (link)). Cultures were grown statically in candle jar atmosphere. As required, cultures were synchronized with 5% (w/v) sorbitol. Asynchronous P. falciparum Dd2 parasites were washed twice in 15 ml incomplete Cytomix (25 mM Hepes, 0.15 mM CaCl2, 5 mM MgCl2, 2 mM EGTA, 120 mM KCl, 5 mM K2HPO4, 5 mM KH2PO4) and resuspended in a total 525 μl with 100 μg of maxi-prep plasmid DNA dissolved in incomplete cytomix (125 μl packed infected red blood cells (iRBCs) and 400 μl DNA/incomplete cytomix). The parasites were transfected in Bio-Rad Gene Pulser cuvette (0.2 cm), 0.31 kV, 950 up, infinity resistance. Selection (10 nM WR99210 or 2 μM DSM-1) was added to parasite 48 h after transfection and used to select resistant parasites (61 (link)). The DNA was isolated from the selected parasite cultures and each cell culture was genotyped for the presence of engineered version of RACK1 variants using PCR genotyping (Fig. S8 ) and subsequent Sanger sequencing (Table S5 ). The rDNA from genomic DNA was used as a control by amplification of 18S rRNA locus (151 bp) by 18S rRNA specific qPCR primers (Fig. S8 and Table S6 ).
7
Targeted FAM122A Gene Deletion
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The sgRNAs targeted to the exon of Fam122a were inserted into the pX330 vector and are listed in Supplementary Table S1 . The plasmids were electroporated into mESCs at 250 V and 500 µF in a 0.4 cm Gene Pulser cuvette (Bio-Rad). Then, mESCs were replated on feeder cells, and individual colonies were picked and expanded. The deletion of FAM122A was validated by Western blot.
8
CRISPR-Cas9 Gene Deletion in E. coli
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Complete deletion of yciS, yciM and both of yciS and yciM were constructed by pEcCas/pEcgRNA system provided by Li Q lab (Li et al., 2021 (link)). Special N20 sequence of each gene was designed using Benchling and fused with BsuI-linearized pTargetF using Golden Gate Assembly to yield target pEcgRNA for yciS and yciM. The electroporation of competent cells of E. coli MG1655 carrying pEcCas was performed using protocol provided by Cha et al.(Cha et al., 1997 (link); Pujol and Kado, 2000 (link); Sharan et al., 2009 (link)). The culture medium was supplemented with a final concentration of 10 mM L-arabinose to induce the expression of λ-Red system. For genome editing, 100 μL of competent cells were mixed with 100 ng of pEcgRNA series plasmids and 400 ng of donor DNA, and the mixture was electroporated in a precooled 1 mm Gene Pulser cuvette (Bio-Rad, Hurcules, USA) at 1.8 kV. The electroporation mixture was immediately suspended in 1 mL of fresh LB medium. Cells were recovered by incubating at 37°C for 1 h before spreading on LB plates containing kanamycin and spectinomycin, and the plates were then incubated overnight at 37°C. Individual colonies were randomly picked and verified by colony-PCR and DNA sequencing. The pEcgRNA series plasmids of verified strains were eliminated by methods provided by Li Q lab (Li et al., 2021 (link)).
9
Electrotransformation of Eubacterium limosum
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Electrotransformation was conducted as previously described [49 (link)]. All of the experiments were carried out in an anaerobic chamber. E. limosum were cultured in 100 mL DSM 135 medium supplemented with 5 g/L glucose. At the mid-exponential phase, the cell pellet was obtained via centrifugation for 15 min at 10,000 g. The collected cell pellet was washed twice with 100 mL sucrose buffer (270 mM) and resuspended to a final concentration of 1011 cells/mL, then transferred to a 0.1-cm-gap Gene Pulser cuvette (Bio-Rad). Then, 1 μg plasmid was added to the prepared cells and pulsed at 0.63 kV. Immediately following, the cells were recovered using 1 mL reinforced clostridial medium (RCM) and incubated at 37 °C until clear growth was observed (6 to 8 h). The recovered cells were plated on an RCM plate (1.5% agar) containing an appropriate antibiotic. A single colony was selected and cultured in DSM 135 medium supplemented with 5 g/L glucose for proliferation, then confirmed via plasmid DNA isolation using a DNA-spin™ plasmid DNA purification kit (iNtRON).
10
Efficient Transfection of L. tarentolae Promastigotes
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Transfections were performed on mid-log phase
L. tarentolae promastigotes. A total of 5 × 10
7 parasites were pelleted (1200xg, 10 min) and washed once with ice-cold PBS before resuspending in 100 μl of transfection buffer (90 mM NaPO
4, 5 mM KCl, 50 mM HEPES, 0.15 mM CaCl
2, pH7.3). The suspension was transferred to a 1 ml Bio-Rad GenePulser® cuvette, and mixed with 10 μg of plasmid pGL1132 DNA in 20 μl sterile water, or 20 μl of sterile water as a negative control. Parasites were electroporated using an Amaxa NucleofectorTM II and subsequently transferred to 10 ml of complete HOMEM in a T25 non-vented flask (Corning). After an overnight incubation at 25°C, appropriate drugs (G418 or Hygromycin, 25 μg/ml) were added in the culture to select for transfectants. Successful transfection was evaluated by confirmation of fluorescence using a Carl Zeiss Axiovert 40 CFL inverted epifluorescence microscope (488 nm excitation and 509 nm emission).
L. tarentolae promastigotes. A total of 5 × 10
7 parasites were pelleted (1200xg, 10 min) and washed once with ice-cold PBS before resuspending in 100 μl of transfection buffer (90 mM NaPO
4, 5 mM KCl, 50 mM HEPES, 0.15 mM CaCl
2, pH7.3). The suspension was transferred to a 1 ml Bio-Rad GenePulser® cuvette, and mixed with 10 μg of plasmid pGL1132 DNA in 20 μl sterile water, or 20 μl of sterile water as a negative control. Parasites were electroporated using an Amaxa NucleofectorTM II and subsequently transferred to 10 ml of complete HOMEM in a T25 non-vented flask (Corning). After an overnight incubation at 25°C, appropriate drugs (G418 or Hygromycin, 25 μg/ml) were added in the culture to select for transfectants. Successful transfection was evaluated by confirmation of fluorescence using a Carl Zeiss Axiovert 40 CFL inverted epifluorescence microscope (488 nm excitation and 509 nm emission).
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