Agroinfiltration was carried out as previously described (Lu et al., 2012 (link)) with some modifications. Briefly, A. tumefaciens C58C1 (pTiB6S3ΔT)H competent cells were transformed with overexpression constructs using an electroporation system (Bio-Rad Laboratories, Hercules, CA, USA). Then, A. tumefaciens was incubated in 10 mL YEB at 28°C until the optical density at 600 nm (OD600) reached 0.8–1.0. After centrifugation (4,000 rpm, 10 min, in room temperature), A. tumefaciens was suspended in 20 mL AB-MES medium (17.2 mM K2HPO4, 8.3 mM NaH2PO4, 18.7 mM NH4Cl, 2 mM KCl, 1.25 mM MgSO4, 100 μM CaCl2, 10 μM FeSO4, 50 mM MES, 2% glucose (w/v), and pH 5.5) with 200 μm acetosyringone and cultured with shaking at 28°C overnight. The overnight culture was centrifuged (4,000 rpm, 10 min, at room temperature), the supernatant was removed, and the pellet of A. tumefaciens was suspended in 2 mL of infiltration medium containing 50% MS medium (1/2 MS salt supplemented with 0.5% sucrose (w/v), pH 5.5), 50% AB-MES and 200 μm acetosyringone. Suspensions of A. tumefaciens cells showing OD600 values of 0.4 and 0.8 were infiltrated into Stages 3 and 4 petals, respectively, by using insulin syringes (60 mm × 31 G).
Electroporation system
Manufactured by Bio-Rad
Sourced in United States
The Electroporation system is a laboratory instrument designed to facilitate the delivery of nucleic acids, such as DNA or RNA, into cells through the application of an electric field. It utilizes a controlled electrical pulse to temporarily increase the permeability of cell membranes, allowing the exogenous material to enter the cells. This system provides a reliable and efficient method for genetic transformation and transfection experiments in various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Panfect reagent, by PAN Biotech (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (2 mentions)
Mir 20a 5p mimic, by GenePharma (1 mentions)
Control mimic and inhibitor, by GenePharma (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Clonexpress 2 one step cloning kit c112 01, by Vazyme (1 mentions)
12 protocols using electroporation system
1
Agroinfiltration Protocol for Transient Gene Expression
2
Overexpression of miR-20a in HCC cells
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The human HCC cell line MHCC97H was cultured in Dulbecco's Modified Eagle's Medium replenished with 10% fetal bovine serum (FBS, Gibco, Life Technologies Limited, Paisley, UK). The human HCC cell line Human hepatocellular carcinoma-7 was cultured in Modified Eagle's Medium containing 10% FBS. All cells were cultured at 37°C in a humidified 5% CO2 incubator. The synthesized miR-20a-5p mimics and the random negative control RNAs (control mimic and inhibitor) were obtained from GenePharma (Shanghai, China). When the cells in the plate reached a fusion degree of 70% to 80% per well, the overexpression of miR-20a was performed by transfecting a miR-20a mimic with a synthesized double-stranded RNA oligonucleotide imitating the miR-20a precursor. The plasmids were transfected into the cells by an electroporation system (Bio-Rad, USA) based on the protocols.
3
Versatile DNA and RNA Transfection Methods
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DNA transfection in HEK293 and HEK293T cells was conducted by the well-established calcium phosphate method, using 25 µg DNA/mL of calcium phosphate solution. DNA transfection in NIH3T3, NCCIT and HepG2 cells was conducted with Lipofectamine 2000 reagent following the manufacturer's instructions (Invitrogen) and using a ratio 1 µg:2 µL DNA:Lipofectamine 2000. DNA transfection in ESCs was conducted with PANfect reagent following the manufacturer's instructions (PAN biotech) and using a ratio 1 µg:2 µL DNA:PANfect. Jurkat E6.1 cells were transfected by electroporation in 1:1 RPMI:FBS media using an exponential wave program in a Bio-Rad electroporation system and providing a single pulse of 240 V. All DNA-transfected cells were harvested 48 h post-transfection (hpt) for further analysis. siRNA/miRNA transfection for all cell lines was conducted with Lipofectamine RNAiMAX reagent following the manufacturer's instructions (Invitrogen) and using a ratio 10 pmol:2 µL RNA:Lipofectamine RNAiMAX. Cells transfected with siRNAs/miRNAs were harvested 72 hpt for further analysis. For experiments requiring siRNA/miRNA and DNA transfections, DNA was transfected 24 h after siRNA/miRNA transfection. MiRNAs and siRNAs used can be found in Supplemental Tables 1 and 2 , respectively.
4
Electroporation of Fusosomes with Nucleic Acids
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Example 21
This example describes electroporation of fusosomes with nucleic acid cargo.
Fusosomes are prepared by any one of the methods described in a previous Example. Approximately 109 fusosomes and 1 μg of nucleic acids, e.g., RNA, are mixed in electroporation buffer (1.15 mM potassium phosphate pH 7.2, 25 mM potassium chloride, 60% iodixanol w/v in water). The fusosomes are electroporated using a single 4 mm cuvette using an electroporation system (BioRad, 165-2081). The fusosomes and nucleic acids are electroporated at 400 V, 125 gF and ∞ ohms, and the cuvette is immediately transferred to ice. After electroporation, fusosomes are washed with PBS, resuspended in PBS, and kept on ice.
See, for example, Kamerkar et al., Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer, Nature, 2017
5
Engineering Threonine-Producing E. coli with Fimbriae Modulation
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Escherichia coli W1688 (CCTCC M2015233) was an L -threonine producer obtained from E. coli MG1655 (ATCC47076) by mutation and molecular modification. It could not produce biofilms apparently. All strains and plasmids used in this work are listed in Tables 1 , 2 , respectively. The fimH gene was amplified from the genomic DNA of E. coli W1688. The fimH gene and plasmid pET28a (with restriction enzyme XbaI and NcoI) were ligated by using the ClonExpress II One Step Cloning Kit C112-01 (Vazyme, Nanjing, China), resulting in a plasmid pET28a-fimH. The final engineered strain was named E. coli W1688-fimH* with Kanamycin resistance for screening. On the other hand, fimH from E. coli W1688 was deleted by Red homologous recombination, resulting in an E. coli W1688-ΔfimH (Madyagol et al., 2011 (link)). Briefly, a PCR-generated Kanamycin resistance marker was used as knock-in DNA fragment. The Kanamycin resistance marker consisted of a Kanamycin resistance sequence in plasmid pKD4 and homologous regions (50–100 bp) flanking the target locus. The knock-in component was transformed into strain E. coli W1688-pKD46 using Bio-Rad electroporation system set at 2.0 kV, 25 mF with a 200 Ohm pulse controller.
6
Modulating EGR1 and NAB2 Expression
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Cells were suspended in serum-free medium, which was followed by transfection with control siRNA (sc-37007) or an siNAB2 mixture (sc-36014) consisting of three specific targeting oligonucleotides (Santa Cruz Biotechnology) using an electroporation system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). For exogenous EGR1 overexpression, the pCDNA3.1-EGR1 vector was used [36 (link)]. The pcMV6-AC-GFP NAB2 vector (Origin Technologies, Rockville, MD, USA) was used for NAB2 overexpression.
7
Cycloheximide Protein Turnover Assay
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10 million MEF or Saos-2 cells were transfected with combinations of constructs by using the BioRad electroporation system. After 36 hours post-transfection, the transfected cells as well as indicated B-cells were incubated with 40 μg/ml cycloheximide (CHX) at specific time points. Then cells were harvested, lysed with RIPA buffer, and analyzed by western blot. Odyssey 3.0 software was used to quantify the band intensities.
8
Transient Transfection of Cancer Cells
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To introduce the destabilized 3’UTR into the cancer cells, we electroporated constructs into 200,000–500,000 cells with 50 ng of plasmid containing the constructs using the BioRad electroporation system. We used the preset mammalian protocol set for 293 T cells and pulsed the cells in a cuvette 2x. The cells were then seeded into six-well plates and viewed for morphology and red fluorescent protein (RFP) expression in 24 hrs. After 24 hrs, the cells expressed RFP indicating that the constructs were successfully integrated into the cells. The constructs were also injected intraperitoneally to the animals as a plasmid.
9
Multimodal Transfection in Cell Lines
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DNA transfection in HEK293 and HEK293T cells was conducted by the well-established calcium phosphate method, using 25 μg DNA/ml of calcium phosphate solution. DNA transfection in NIH3T3 and HepG2 cells was conducted with Lipofectamine 2000 reagent following the manufacturer's instructions (Invitrogen) and using a ratio 1μg:2μL DNA:Lipofectamine 2000. DNA transfection in ESCs was conducted with PANfect reagent following the manufacturer's instructions (PAN biotech) and using a ratio 1μg:2μL DNA:PANfect. Jurkat E.6 cells were transfected by electroporation in 1:1 RPMI:FBS media using an exponential wave program in a Bio-Rad electroporation system and providing a single pulse of 240 V. All DNA-transfected cells were harvested 48 h post-transfection (hpt) for further analysis. siRNA/miRNA transfection for all cell lines was conducted with Lipofectamine RNAiMAX reagent following the manufacturer's instructions (Invitrogen) and using a ratio 10pmol:2μL RNA:Lipofectamine RNAiMAX. Cells transfected with siRNAs/miRNAs were harvested 72 hpt for further analysis. For experiments requiring siRNA/miRNA and DNA transfections, DNA was transfected 24 h after siRNA/miRNA transfection. MiRNAs and siRNAs used can be found in Suppl. Materials Tables 1 and2, respectively.
10
Plasmid Cloning and Verification Protocol
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A long fragment primer was designed to extend the target fragment (Table 1). The primer was designed according to Datsenko. PCR amplification used pKD3 plasmid as templates. The PCR product was purified by gel recovery, digested with DpnI, and then re-purified again. Finally, the recovered products were dissolved in water. The concentrations of the PCR products after purification were required to exceed 100 ng/μL. The pKD46 plasmid was cloned into the E. coli K-12 strain and detected with primers (Table 2). Then 5 μL of target fragment and 50 μL of electroconversion-sensing cells were added to a 0.2 cm chamber and subjected to electric shock with a Bio-Rad electroporation system. After the electric transfer, the product was coated on the corresponding resistance plate for screening after incubation at room temperature overnight. Positive clones on the plate were selected for PCR verification. Two pairs of primers were designed, and four pairs of primers were cross-checked for PCR verification (Table 1). The lengths of the four pairs of primer amplification fragments from the positive strain differed from those of the strain without knockout. PCR positive strains were sent to Shanghai Biological Engineering Co., Ltd for sequencing verification.
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