Btx ecm 830 electroporator

Manufactured by Harvard Apparatus

Sourced in United States

The BTX ECM 830 electroporator is a laboratory instrument used for the introduction of foreign molecules, such as DNA, RNA, or other macromolecules, into cells through the process of electroporation. The device generates and delivers controlled electrical pulses to samples, temporarily increasing the permeability of cell membranes to facilitate the uptake of the desired materials.

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Lab products found in correlation

Fast green, by Merck Group (6 mentions) 5 fluorodeoxyuracil, by Merck Group (3 mentions) Fast green dye, by Merck Group (3 mentions) Picospritzer 2, by Parker Hannifin (2 mentions) Chloramphenicol, by Merck Group (2 mentions) Pv820 pneumatic picopump, by World Precision Instruments (1 mentions) Rompun 2, by Bayer (1 mentions) Femtojet injector, by Eppendorf (1 mentions) Pc 100, by Narishige (1 mentions) Pulled glass capillaries, by Harvard Apparatus (1 mentions) Diclofenac, by Novartis (1 mentions) Voltaren, by Novartis (1 mentions) Mpa xa, by Merck Group (1 mentions) Dna sequencing, by Thermo Fisher Scientific (1 mentions) Lab tek 2, by Thermo Fisher Scientific (1 mentions) Mem a, by Thermo Fisher Scientific (1 mentions) Rat tail collagen type 1, by BD (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Pulled glass micropipette, by Drummond (1 mentions) Pgpu6 gfp neo vector, by GenePharma (1 mentions)

38 protocols using btx ecm 830 electroporator

Knockdown of Genes in Adult Zebrafish

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Microinjection and electroporation of morpholino oligonucleotides (MOs; Gene-Tools, LLC, Philomath, OR) was used as described for knockdown experiments in adult zebrafish [20 (link)–22 (link)]. Briefly, lissamine-tagged MOs (~0.2 μL, 1 mM in nuclease-free H₂O) were directly microinjected into the LR muscle followed by electroporation (6 to 10 pulses at 48 V/cm, BTX ECM830 electroporator; Harvard Apparatus, Holliston, MA). Microinjections were performed 3 h prior to LR injury, and MO uptake was confirmed via lissamine fluorescence prior to myectomy. MOs were designed to target the 5′-UTR of respective mRNAs (translational blocking MOs), and were compared to a standard control MO (CON) targeting a human beta-globin intron mutation. When possible, previously published MOs were utilized (Table 3). For all others, MO sequence design was performed as a service by Gene Tools. All MOs were injected at the same concentration, and all experiments were performed using 5 fish per experimental group per time point, unless stated otherwise in the text and/or figure legend. No significant mortality was noted.Table 3

Sequence of the morpholino antisense oligonucleotides (MOs)

	Sequence	Reference
ezh2	5′-CGATTTCCTCCCGGTCAATCCCATG	[62 (link)]
fn1a	5′-TTTTTTCACAGGTGCGATTGAACAC	[63 (link)]
suz12a	5′-GAGCCATCCTAAAATAGCGTTCGTG	[64 (link)]
Control (CON)	5′-CCTCTTACCTCAGTTACAATTTATA	[65 (link)]

Louie K.W., Saera-Vila A., Kish P.E., Colacino J.A, & Kahana A. (2017). Temporally distinct transcriptional regulation of myocyte dedifferentiation and Myofiber growth during muscle regeneration. BMC Genomics, 18, 854.

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In utero Electroporation of Embryonic Mouse Brains

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In utero electroporation was performed as described previously [59 (link)]. Briefly, timed pregnant female mice from E14.5 of gestation were deeply anesthetized, and the uterine horns were gently exposed by laparotomy. The lateral ventricles of an embryonic brain were injected with plasmid DNA (2 μg/μl) and 0.001% fast green (Sigma-Aldrich) using a Picospritzer II (Parker Hannifin). Electroporation was achieved by placing two sterile forceps-type electrodes on opposing sides of the uterine sac around the embryonic head and applying a series of short electrical pulses using a BTX ECM 830 electroporator (Harvard Apparatus) (five pulses with 100 ms length separated by 900 ms intervals were applied at 45 V).

Ka M., Kim H.G, & Kim W.Y. (2022). WDR5-HOTTIP histone modifying complex regulates neural migration and dendrite polarity of pyramidal neurons via reelin signaling. Molecular neurobiology, 59(8), 5104-5120.

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Transient Transfection of Parasites

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Following natural egress, freshly harvested parasites were transfected with plasmids, using protocols previously described (87 (link)). In brief, ~2 × 107 extracellular parasites were resuspended in 370 μL cytomix buffer were mixed with ~30 μL purified plasmid or amplicon DNA in a 4-mm gap BTX cuvette and electroporated using a BTX ECM 830 electroporator (Harvard Apparatus) using the following parameters: 1,700 V, 176-μs pulse length, 2 pulses, 100-ms interval between pulses. Transgenic parasites were isolated by outgrowth under selection with mycophenolic acid (25 μg/mL) and xanthine (50 μg/mL) (MPA/Xa), pyrimethamine (Pyr) (3 mM), chloramphenicol (40 mM), 5-fluorodeoxyuracil (10 μM) (Sigma), Phleomycin (Phleo) (5 μg/ml) (InvivoGen) as needed. Stable clones were isolated by limiting dilution on HFF monolayers grown in 96-well plates (Figure S3).

Henry B., Sibley L.D, & Rosenberg A. (2023). A Combination of Four Nuclear Targeted Effectors Protects Toxoplasma Against Interferon Gamma Driven Human Host Cell Death During Acute Infection. bioRxiv.

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In Utero Neuronal Migration Manipulation

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In utero injections and electroporations were performed as previously described (66) in embryos from timed pregnant rats (embryonic day 15) that were anaesthetized with ketamine (100 mg/kg, IMALGENE 1000; Merial, Lyon, France) / xylazine (10 mg/kg, Rompun 2%; Bayer Healthcare, Leverkusen, Germany). Wistar rats (Janvier, Le Genest-Saint-Isle, France) were raised and mated at INMED Post Genomic Platform (PPGI) animal facility in agreement with the European Union and French legislations. The uterine horns were exposed, and a lateral ventricle of each embryo was injected using pulled glass capillaries and a microinjector (PV 820 Pneumatic PicoPump; World Precision Instruments, Sarasota, FL) with Fast Green (2 mg/mL; Sigma, St Louis, MO, USA) combined with the constructs encoding Cl-Sensor plus scrambled shRNA, or WNK1 shRNA (ratio 1:3).
Plasmids were further electroporated by discharging a 4000 µF capacitor charged to 40 V with a BTX ECM 830 electroporator (BTX Harvard Apparatus, Holliston, MA, USA). The voltage was discharged in five electrical pulses at 950 ms intervals via tweezer-type electrodes placed on the head of the embryo across the uterine wall. We performed in utero electroporation in embryonic rats at E15, corresponding to an active period of both radial and tangential migration of newborn neurons in the cortex.

Friedel P., Kahle K.T., Zhang J., Hertz N., Pisella L.I., Buhler E., Schaller F., Duan J., Khanna A.R., Bishop P.N., Shokat K.M, & Medina I. (2015). WNK1-regulated inhibitory phosphorylation of the KCC2 cotransporter maintains the depolarizing action of GABA in immature neurons. Science signaling, 8(383).

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In Utero Electroporation of Rat Embryos

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E16.5 Sprague-Dawley embryos were injected and electroporated in utero. Timed-pregnant E16.5 females were anesthetized with isoflurane after a preoperative dose of buprenorphine (0.05 mg/kg), 15 min before surgery. The abdomen was cut, and the uterine horn was exposed. One microliter of DNA (0.5 μg/μl for shRNA Prickle2 and 0.25 μg/μl for control plasmid) prepared in endo-free water mixed with 1% Fast-Green dye (Sigma-Aldrich) was injected through the uterine wall into the lateral ventricle of each embryo using pulled glass capillaries (Harvard Apparatus). Capillaries were prepared using a needle pipette puller (Narishige, PC-100), and injection was performed with a Femtojet injector (Eppendorf). For electroporation, five 50-ms pulses at 55 V with 950-ms breaks were delivered through the embryonic brain to target the cortex using 7-mm electrode paddles connected to a BTX ECM830 electroporator (Harvard Apparatus). After the procedure, the abdomen was filled with a 37°C-warmed PBS solution, and the wound was closed using surgical suture. Animals were put back in their home cage in the animal facility. Rats gave birth at E22.5, and the pups were kept with their mother until collection at P5 or P6.

Dorrego-Rivas A., Ezan J., Moreau M.M., Poirault-Chassac S., Aubailly N., De Neve J., Blanchard C., Castets F., Fréal A., Battefeld A., Sans N, & Montcouquiol M. (2022). The core PCP protein Prickle2 regulates axon number and AIS maturation by binding to AnkG and modulating microtubule bundling. Science Advances, 8(36), eabo6333.

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Genetic Modification of Parasites

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Parasites were harvested and resuspended in Cytomix (10 mM KPO₄, 120 mM KCl, 5 mM MgCl₂, 25 mM HEPES, 2 mM EDTA) 1 × 10⁸ parasites/ml, then combined with 10–20 μg pCas9-sgRNA 3′ and 1–5 μg amplicons with 2 mM ATP, 2 mM GSH, and 150 μM CaCl₂ to a final volume of 250 µl, followed by electroporation using a BTX ECM 830 electroporator (Harvard Apparatus), as described previously (Soldati and Boothroyd, 1993 (link)). Parasites were selected on the second day with addition of appropriate drugs mycophenolic acid (MPA) (25 µg/ml) and 6-xanthine (6Xa) (50 µg/ml), or pyrimethamine (Pyr) (3 µM). Negative selection was applied with 6-thio-xanthine (200 μg/ml) when parasites were transfected with a pCre plasmid to remove the resistant marker HXGPRT. Positive selection pools of parasites were subcloned in 96-well plates at 3 parasites/well and allowed to grow for 6–7 days before selection of wells containing a single plaque. Pure clones were diagnosed by PCR using appropriate primers (Supplementary file 1d) and IFA analysis using appropriate antibodies if applicable.

Dong H., Yang J., He K., Zheng W.B., Lai D.H., Liu J., Ding H.Y., Wu R.B., Brown K.M., Hide G., Lun Z.R., Zhu X.Q, & Long S. (2024). The Toxoplasma monocarboxylate transporters are involved in the metabolism within the apicoplast and are linked to parasite survival. eLife, 12, RP88866.

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In Vivo Lmnb1 Silencing by Electroporation

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Lmnb1 silencing in vivo was carried out by in utero electroporation using Lmnb1 shRNA (Sh-Lmnb1) or scramble-shRNA (Sh-Scr) plasmids as described^{35 (link)}. The pregnant female C57BL6/J mouse (E14) was anesthetized using isofluorane. After injection of DNA (3 µg) containing 1% Fast-Green dye (Sigma) through the uterine wall into the lumen of the telencephalic vesicles, each embryo was subjected to electroporation by five square electric pulses (30 mV for 50 ms, 1 s intervals) delivered through platinum electrodes (Sonidel) using a BTX-ECM 830 electroporator (Harvard Apparatus). After electroporation, the abdominal cavity was sutured and the pregnant mouse was administered diclofenac (0.5 mg/kg) (Voltaren, Novartis) by intraperitoneal injection and kept warm until awake. For Lmnb1 knockdown studies, electroporated pups were born and immediately transferred to a foster CD-1 mother. The pups were sacrificed at postnatal day 7 (P7) and the brains collected for analysis.

Mahajani S., Giacomini C., Marinaro F., De Pietri Tonelli D., Contestabile A, & Gasparini L. (2017). Lamin B1 levels modulate differentiation into neurons during embryonic corticogenesis. Scientific Reports, 7, 4897.

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In utero Electroporation of miRNA and Fmr1 in Mice

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As previously described^{23 (link)}, miRNA or Fmr1 overexpression constructs or Fmr1 shRNA constructs were electroporated in utero into timed pregnant CD-1 mice. Briefly, E12.5 or E14.5 pregnant dams were anaesthetised using pentobarbital sodium, and the uterine horns were exposed. Two to 3 μg/μl of plasmids or 40 µM miRNA antagomirs spiked with Fast Green (Sigma, Louis, Missouri, USA) were injected into the lateral ventricle of the embryo brain. Electroporation was conducted with electric pulses of 20–30 V for 50 ms, which were repeated five times with 950-ms intervals using the BTX-ECM830 electroporator (Harvard Apparatus, Holliston, Massachusetts, USA).

Wu C., Zhang X., Chen P., Ruan X., Liu W., Li Y., Sun C., Hou L., Yin B., Qiang B., Shu P, & Peng X. (2019). MicroRNA-129 modulates neuronal migration by targeting Fmr1 in the developing mouse cortex. Cell Death & Disease, 10(4), 287.

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Morpholino Knockdown Experiments in Adult Zebrafish

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Microinjection of morpholino oligonucleotides (MOs; Gene-Tools, LLC, Philomath, OR), a widely used technique to perform knockdown experiments in adult zebrafish [29 –31 ], was used. Briefly, lissamine-tagged MOs were directly microinjected into the right LR muscle followed by square-wave electroporation (6 to 10 pulses at 48 V/cm, BTX ECM830 electroporator; Harvard Apparatus, Holliston, MA). Microinjections were performed 3 hours prior to LR injury, and MO uptake was confirmed via lissamine fluorescence prior to myectomy. The MO sequence for igf1ra and igf1rb genes was previously validated in zebrafish [32 , 33 (link)], and a standard MO targeting a human beta-globin intron mutation was used as control. Experiments were performed using 5 fish per experimental group, unless stated otherwise in the text and/or figure legend. No mortality was detected during the experimental process.

Saera-Vila A., Louie K.W., Sha C., Kelly R.M., Kish P.E, & Kahana A. (2018). Extraocular muscle regeneration in zebrafish requires late signals from Insulin-like growth factors. PLoS ONE, 13(2), e0192214.

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Parasites Transfection and Selection

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Following natural egress, freshly harvested parasites were transfected with plasmids, using protocols previously described (Shen et al., 2014 (link)). In brief, ~2 × 10⁷ extracellular parasites were resuspended in 370 μL cytomix buffer were mixed with ~30 μL purified plasmid or amplicon DNA in a 4-mm gap BTX cuvette and electroporated using a BTX ECM 830 electroporator (Harvard Apparatus) using the following parameters: 1,700 V, 176-μs pulse length, 2 pulses, 100-ms interval between pulses. Transgenic parasites were isolated by outgrowth under selection with mycophenolic acid (25 μg/mL) and xanthine (50 μg/mL) (MPA/Xa), pyrimethamine (Pyr) (3 mM), chloramphenicol (20 mM), 5-fluorodeoxyuracil (10 μM) (Sigma), as needed. Stable clones were isolated by limiting dilution on HFF monolayers grown in 96-well plates.

Rosenberg A, & Sibley L.D. (2021). Toxoplasma gondii secreted effectors co-opt host repressor complexes to inhibit necroptosis. Cell host & microbe, 29(7), 1186-1198.e8.

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