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Electroporator

Manufactured by Eppendorf
Sourced in Germany, United States

The Eppendorf Electroporator is a device used for the electroporation of cells, a technique that introduces foreign molecules into cells by creating temporary pores in their membranes using electrical pulses. The device allows users to set and control the electrical parameters, such as voltage, pulse duration, and number of pulses, to optimize the electroporation process for different cell types and applications.

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13 protocols using electroporator

1

Heterologous expression of epothilone in M. xanthus

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The constructed expression plasmids were introduced into M. xanthus cells by electroporation. Briefly, M. xanthus cells from 50-mL overnight cultures (OD600 was about 0.6) were collected and washed thrice with ice-cold water for preparing competent cells. 1.5 mL of the culture was centrifuged and the precipitate was resuspended with 50 μL of ice-cold water in tube, mixed with 3 μg of DNA, and electroporated at a voltage of 1,250 V in a 2 mm cuvette using the Electroporator (Eppendorf, Germany). Then the cells were transferred into 2 mL CYE liquid medium in a 10-mL tube and incubated at 30°C with a rotate speed of 250 rpm. After 4–6 h of incubation, 0.1–0.5 mL of cells was added to 2 mL 0.5% soft agar and the mixture was spread on 1.5% CYE selection agar plates. Resistant colonies that appeared after 6 days of incubation were checked by colony PCR with the following primers: primer pairs of CM-APH and CM-EPOP were used to check the integration of cat gene; Epothilone-specific primers, EA, EP, EB, EC1, EC2, EE and EF, with a 51% coverage of the whole gene cluster, were designed to detect the regions located in ORFs epoA,epoP, epoB, epoC, epoE or epoF, respectively, to verify the integration of the whole biosynthetic gene cluster in the M. xanthus chromosome.
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2

Genetic Transformation of Sugary-1 Mutant Rice

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Genomic DNA fragments (8.6 kb) containing Sobic.007G204600 from MS3B and SUF were introduced into pCAMBIA1300 (Cambia) using the KpnI/EcoRI sites. Each construct was transformed into Agrobacterium tumefaciens EHA105 using an electroporator (Eppendorf) at 2,500 V and used to infect calli of sugary-1 mutant (SG0807), whose genetic background is Oryza sativa (L.) cv. ‘Kinmaze’ as described by Ozawa (Ozawa, 2012 (link)). Transformed calli and plants were selected by hygromycin resistance, with regenerants grown in pots in a greenhouse.
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3

Recombinant BCG Vaccine Production

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The recombinant plasmid pMV361- EgG1Y162 was transformed into the BCG by electroporation (Electroporator, Eppendorf). The transformed bacteria were cultured for 3 to 4 weeks at 37°C on Middlebrook 7H10 broth agar (BD), containing 10% oleic acid albumin-dextrose-catalase (OADC) and 0.5% Tween 80 in kanamycin selective medium (kanamycin: 30 μg/ml). The transformed bacteria were selected and amplified in the Middlebrook 7H9 liquid medium for 2 to 3 weeks. The number of cells used for vaccination was determined by the colony forming unit (cfu). The method for calculating BCG was based on previous study.16 (link) When A600 is 0.1, the cfu of the BCG or rBCG is approximately 2.5 × 106 cfu. The cells were centrifuged at 3000 rpm for 10 minutes, and diluted to 1 × 106 cfu in 100 μl PBS.
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4

Fluorinated Tyrosine Incorporation in GFP

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BL21(DE3) cells were co-transformed with a pET-GFP-TAG-66 plasmid (0.5 μL) and pEVOL-3FY plasmid (0.5 μL) using an Eppendorf electroporator (Eppendorf, Hauppague, NY, USA). Cells were then plated on LB-agar plates supplemented with ampicillin (50 mg/mL) and chloramphenicol (34 mg/mL) and grown at 37 °C. After 16 h, a single colony was selected and used to inoculate LB media (4 mL) supplemented with ampicillin and chloramphenicol. The culture was grown at 37 °C for 12 h. The culture was used to initiate an expression culture of LB media (10 mL) at OD600 0.1, then incubated at 37 °C, to an OD600 of ~0.6, at which point cells were induced with 1 M IPTG (10 μL), 20% arabinose (10 μL) and 100 mM (100 μL) of respective fluorotyrosine (3-FY; 2,3-F2Y;3,5-F2Y; 2,3,5-F3Y; 2,3,6-F3Y). Cultures were grown for an additional 16 h at 37 °C, then harvested by centrifugation (10 min at 10,000 rpm). The media was removed and the cell pellet placed in the −80 °C freezer for at 20 min. Purification was accomplished using commercially available Ni-NTA spin columns and according to manufacturer’s protocol. Protein yield and purity was assessed by SDS-PAGE, and with a Nanodrop spectrophotometer (ThermoFisher, Pittsburgh, PA, USA).
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5

Fluorotyrosine Incorporation in GFP

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BL21(DE3) cells were co-transformed with a pET-GFP-TAG-66 plasmid (0.5 μL) and pEVOL-3FY plasmid (0.5 μL) using an Eppendorf electroporator (Eppendorf, Hauppague, NY, USA). Cells were then plated on LB-agar plates supplemented with ampicillin (50 mg/mL) and chloramphenicol (34 mg/mL) and grown at 37 °C. After 16 h, a single colony was selected and used to inoculate LB media (4 mL) supplemented with ampicillin and chloramphenicol. The culture was grown at 37 °C for 12 h. The culture was used to initiate an expression culture of LB media (10 mL) at OD600 0.1, then incubated at 37 °C, to an OD600 of ~0.6, at which point cells were induced with 1 M IPTG (10 μL), 20% arabinose (10 μL) and 100 mM (100 μL) of respective fluorotyrosine (3-FY; 2,3-F2Y; 3,5-F2Y; 2,3,5-F3Y; 2,3,6-F3Y). Cultures were grown for an additional 16 h at 37 °C, then harvested by centrifugation (10 min at 10,000 rpm). The media was removed and the cell pellet placed in the −80 °C freezer for at 20 min. Purification was accomplished using commercially available Ni-NTA spin columns and according to manufacturer’s protocol. Protein yield and purity was assessed by SDS-PAGE, and with a Nanodrop spectrophotometer (ThermoFisher, Pittsburgh, PA, USA).
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6

Expression and Purification of cHSPA6

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The ORF of cHSPA6 cloned on pET15 vector and expression host E. coli BL21 (DE3) pLysS were kindly provided by Elrobh et al. (2011) (link). IPTG and ampicillin were obtained from Biobasic. Benzonase was purchased from Sigma, Chicken egg lysozyme from USB Corporation. Superdex 75, Ni–NTA resin, low molecular weight markers and prepacked columns were from Amersham Biosciences. All other chemicals used in this study were of reagent grade. Ultrospec 2100 pro Spectrophotometer, AKTA purification system, SDS–PAGE assembly were from Amersham Biosciences. Thermomixer, electroporator and benchtop cooling centrifuge were from Eppendorf. Lamp sterilizer from Cole-Parmer, shaking incubator from Jeio Tech, South Korea, gel scanner from Epson and pH meter was from Sentron.
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7

Construction and Screening of Aa113 BAC Library

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The Aa113 genomic DNA bacterial artificial chromosome (BAC) library was constructed as described by Brosch et al. (1998) (link) with minor modifications. After HindIII partial digestion, DNA fragments 75 to 120 kb in size were ligated into plasmid pBeloBAC11 and electroporated into E. coli TG1 using an electroporator (Eppendorf, Germany). After electroporation, the cells were resuspended in 600 µl of LB medium (10 g tryptone, 5 g yeast extract, and 10 g NaCl in 1000 ml deionized water), allowed to recover for 1 h at 37° with gentle shaking, and then plated on LB agar containing chloramphenicol (12.5 μg/ml). The plates were incubated overnight, and the recombinants were picked manually and transferred to 96-well plates that were incubated for 10 h with shaking at 220 rpm. The BAC library was stored at −80°. Screen of the BAC clone by pool method was first performed with the markers SC403 and SC254. Gaps present in the assembled sequence were further filled as described by Zhou et al. (2006) (link). The selected BAC clones were sequenced, and the obtained sequences were assembled using the Bioedit software (http://www.mbio.ncsu.edu/bioedit/bioedit.html).
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8

Somatic Cell Nuclear Transfer in Oocytes

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Trypsinized and washed donor cells were transferred into the perivitelline spaces of enucleated oocytes with a 25-μm micropipette. Cell couplets were briefly washed in fusion medium (0.3 M mannitol, 0.1 mM MgSO4, 0.05 mM CaCl2, 0.05% fatty acid-free BSA) and fused by two DC pulses of 100V for 15 μs each using an Eppendorf electroporator. Couplets were removed from the fusion chamber and put back into Hepes-TCM-199 to score fusion success and detect detached or lysed donor cells. Reconstructs were activated 1.5 h post fusion with 5 μM ionomycine followed by exposure to 6-dimethylaminopurine (6-DMAP) for 4 h, as described previously [14 (link)]. The activated oocytes were then transferred to 500 μL of embryo culture medium I (modified potassium simplex optimization medium with essential and non-essential amino acids [KSOMaa] supplemented with 1% BSA and cultured at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 in air. On Day 3 (Day 0 = day of activation) the cleaved embryos were transferred into 500 μL of embryo culture medium II (modified KSOMaa supplemented with 10% FCS) and cultured under the same conditions until Day 7. The proportion of oocytes that cleaved was recorded on Day 3, and those that reached morula and blastocyst stages were recorded on Day 7 of culture.
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9

Plasmid Transformation of M. extorquens AM1

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The plasmids were transformed into M. extorquens AM1 by electroporation as described in a previous study [53 (link)], with a few adjustments. Initially, M. extorquens AM1 cells were grown in a 50-mL flask until the culture reached OD600 of 0.8. The cells were then collected, washed thrice with ice-cold water to prepare competent cells, and resuspended in 1 mL ddH2O for distribution. Next, 100 µL of competent cells mixed with 2 µg of plasmid DNA were transferred into a 1 mm-gap cuvette placed on ice and operated at a voltage of 1800 V using the electroporator (Eppendorf, Germany). The mixture was transferred into 2 mL liquid medium in a 10 mL tube and incubated at 30℃ with a rotational speed of 200 rpm for 5 h. 100 µL of the culture was subsequently spread onto selective plates. The objective colonies appeared after 3–5 days via antibiotic resistance, which were confirmed by colony PCR.
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10

Optimizing GNPs Uptake in HT-29 Cells

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4 × 106 cell /ml HT-29 colon cancer cells were trypsinized, centrifuged and suspended in the RPMI 1640 medium. Then,
400 µl cell suspensions incubated with 60 µM of GNPs and was added into a sterile electroporation cuvette (Eppendorf, Netherlands) with a 4-mm gap between the electrodes.
The cell/GNPs mixture of the cuvette was incubated for 10 min at 4 °C on ice and then transferred to an electroporation chamber.
Number of four electric pulses of different voltages 200, 400, 600, 800, 1000 and 1200 with duration of 100 µs and pulse number
of 2 were delivered using an Electroporator (Eppendorf). Field amplitudes higher than 1200 V/cm are not normally tested as it is known that they are highly
toxic for the cells (induction of irreversible electroporation and, thus, of cell death). Immediately after electroporation, the cuvette was taken
out from the chamber and incubated for 2 min at 4 °C on ice and then cells were stored at humidified atmosphere at 37 °C with 5% CO2for 3-4 hr before viability and uptake assay processes using GFAAS analysis as described above.
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