E z n a plasmid dna mini kit 1

Manufactured by Omega Bio-Tek

Sourced in United States

The E.Z.N.A Plasmid DNA Mini Kit is a laboratory product that provides a simple and efficient method for the isolation of high-quality plasmid DNA from bacterial cultures. The kit utilizes a silica-based membrane technology to capture and purify plasmid DNA, which can then be eluted for use in downstream applications.

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Lab products found in correlation

Hifi dna assembly, by New England Biolabs (1 mentions) Kld enzyme mix, by New England Biolabs (1 mentions) Gel imaging system, by Azure Biosystems (1 mentions) Hotstart q5, by New England Biolabs (1 mentions) Pgem t easy vector system, by Promega (1 mentions) Nanodrop 1000, by Thermo Fisher Scientific (1 mentions) Escherichia coli jm 109 cells, by Promega (1 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions) Hindiii, by Thermo Fisher Scientific (1 mentions) Primestar hs dna polymerase, by Takara Bio (1 mentions) E z n a gel extraction kit, by Omega Bio-Tek (1 mentions) Restriction endonuclease, by New England Biolabs (1 mentions) Nebuilder hifi dna assembly master mix, by New England Biolabs (1 mentions) Wizard dna purification kit, by Promega (1 mentions) Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Plasmid Mini Kit (30 citations) Mini Kit I (2 citations)

8 protocols using e z n a plasmid dna mini kit 1

Rapid Plasmid Cloning in V. natriegens

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In order to test the utility of NPT and V. natriegens as a host for cloning, we designed two arbitrary Gibson assembly and KLD reactions, creating final plasmids pDS5.43 and pDS5.44, respectively. The requisite PCRs were completed using NEB Hot Start Q5. PCR product for Gibson assembly was cleaned using a Zymo Clean & Concentrator kit and then used in a NEBuilder HiFi DNA Assembly reaction as described by the manufacturer.
In the KLD reaction (NEB KLD Enzyme Mix), pDS5.30 is PCR amplified using primers TransientKan_F and DelGFP_R in order to excise the GFP sequence from pDS5.30, producing plasmid pDS5.44 and an easy-to-visualize change from green to white cells. This PCR product is used directly in the KLD reaction without further cleaning, per the manufacturer’s instruction. In both, 2 μL of reaction product is added directly to the competent cells in media, just as with plasmid DNA in the previously described NPT protocol.
Miniprep extraction of plasmid DNA from V. natriegens was accomplished using the E.Z.N.A Plasmid DNA Mini Kit I produced by Omega Bio-Tek, following the manufacturer’s instructions.
For the demonstration of producing single colonies within a standard workday, colonies are imaged using an Azure Biosystems Gel Imaging System. The image contrast is altered in order to highlight the presence of colonies and facilitate measurement of their size.

Specht D.A., Sheppard T.J., Kennedy F., Li S., Gadikota G, & Barstow B. (2024). Efficient natural plasmid transformation of Vibrio natriegens enables zero-capital molecular biology. PNAS Nexus, 3(2), pgad444.

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Viral RNA Extraction and RT-PCR for ARV Detection

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Viral RNA was extracted from the ARV isolate in LMH cell culture fluid using an RNeasy Mini Kit (Cat. No. Z74106, QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. The RT-PCR reaction was performed using a One Step RT-PCR Kit (Cat. No. 210212, QIAGEN, Valencia, CA, USA) with two degenerate primers P1 and P4, corresponding to the σC gene of ARV20 (link). The RT-PCR products were obtained through 1% agarose gel electrophoresis and were purified using a gel extraction kit (Cat. No. 04113KE1, Axygen, Tewksbury, MA, USA) according to the manufacturer’s instructions. Purified RT-PCR product was cloned into pGEM-T easy vector using pGEM-T easy Vector system (Promega, Madison, WI, USA), and then transformed into competent Escherichia coli JM109 cells (Promega, Madison, WI, USA) in accordance with the manufacturer’s instructions. The recombinant plasmids from positive colonies were isolated by using E.Z.N.A.^® Plasmid DNA Mini Kit I (Omega Bio-tek, Norcross, GA, USA) per the manufacturer’s instructions. The concentration of the purified RT-PCR product and recombinant plasmids were obtained using a NanoDrop™1000 (Thermo Scientific, Waltham, MA, USA) spectrophotometer and were subsequently submitted to the Penn State Genomics Core Facility for Sanger sequencing.

Tang Y., Lin L., Sebastian A, & Lu H. (2016). Detection and characterization of two co-infection variant strains of avian orthoreovirus (ARV) in young layer chickens using next-generation sequencing (NGS). Scientific Reports, 6, 24519.

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Overexpression of human eIF4AI in E. coli

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The human eIF4AI cDNA (gene accession number NM 001416.4) under the control of CMV-promotor in a pcDNA3.1(+) vector was acquired under the project ID: U533ZGE280-1, clone ID: HP4425A (GenScript, Piscataway, NJ, USA). Plasmid DNA was transformed into E. coli strain DH5α. Scrapings from colonies were used to inoculate 5 mL of culture medium LB containing 100 μg/mL ampicillin; these mixtures were grown overnight at 37 °C with agitation. Plasmids were purified from the resulting cultures using E.Z.N.A. Plasmid DNA Mini Kit I (Omega Bio-TEK) according to the manufacturer’s protocol and verified through digestion with restriction enzymes HindIII (GibcoBRL, MD) and BamHI (BRL, MD). The purity and yield of DNA were determined by Nanodrop analysis. Transfections were performed using Lipofectamine 3000 (Invitrogen) following the manufacturer’s instructions. Controls were evaluated by WB employing eIF4A monoclonal antibody (ID 2490). The effect of treatments with Dox (500 nM) and Cry (25 µM) was performed.

González-Ortiz A., Pulido-Capiz A., Castañeda-Sánchez C.Y., Ibarra-López E., Galindo-Hernández O., Calderón-Fernández M.A., López-Cossio L.Y., Díaz-Molina R., Chimal-Vega B., Serafín-Higuera N., Córdova-Guerrero I, & García-González V. (2022). eIF4A/PDCD4 Pathway, a Factor for Doxorubicin Chemoresistance in a Triple-Negative Breast Cancer Cell Model. Cells, 11(24), 4069.

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Lentiviral shRNA Library Generation

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shRNA libraries used were based on the pLKO.1-puro TRC vector backbone developed by The RNAi Consortium (TRC). TRC1, TRC1.5, or TRC2 clones from the Sigma Mission TRC - Human collection (Sigma Aldrich) were used, cherry-picked, and obtained as bacterial glycerol stocks from the RNAi center at the La Jolla Institute for Allergy and Immunology (for clone list, see Additional file 8: Table S7). Bacteria were inoculated in overnight cultures and pLKO.1 plasmids were extracted individually with the E.Z.N.A. Plasmid DNA Mini Kit I (Omega Bio-tek) according to the manufacturer’s instructions with an optional second washing step and eluted in sterile 10 mM Tris at pH 8.0. Empty pLKO.1 vector or pLKO.1-Scr (with scrambled non-targeting shRNA) were used as negative controls, and pLKO3G (encoding for GFP in place of puromycin resistance gene) was used to control for transfection and transduction efficiency. A pool of clones targeting human CD4 was used to optimize T cell transduction and knockdown. For knockdown of ‘iTreg candidate genes’ , a pool of 2 to 8 (mean 4.3, SD 0.9) shRNA-encoding plasmids per gene was transfected to 293T cells in equal amounts to produce lentivirus. For some genes, two independent pools of shRNA-encoding plasmids were used. Each pool contained only shRNA-encoding plasmids from the same TRC version (for list of clones, see Additional file 8: Table S7).

Schmidt A., Marabita F., Kiani N.A., Gross C.C., Johansson H.J., Éliás S., Rautio S., Eriksson M., Fernandes S.J., Silberberg G., Ullah U., Bhatia U., Lähdesmäki H., Lehtiö J., Gomez-Cabrero D., Wiendl H., Lahesmaa R, & Tegnér J. (2018). Time-resolved transcriptome and proteome landscape of human regulatory T cell (Treg) differentiation reveals novel regulators of FOXP3. BMC Biology, 16, 47.

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Streamlined Molecular Cloning Protocol

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Chemical reagents, biochemicals, and media components used in this study were purchased from Thermo Fisher Scientific Co. Ltd. (USA) unless otherwise stated. Restriction endonucleases were purchased from New England Biolabs, Inc. (USA). PrimeSTAR HS DNA polymerase (Takara Biotechnology Co., Ltd. Japan) was used for all PCR amplifications on an Eppendorf^® Mastercycler^® Nexus X2 Thermal Cycler (Eppendorf Co., Ltd. Germany). PCR products were purified using the E.Z.N.A.^® Gel Extraction Kit (Omega Bio-tek, Inc., USA). The NEBuilder^® HiFi DNA Assembly master mix (New England Biolabs, Inc., USA) was applied for Gibson assembly. All plasmids were extracted using the E.Z.N.A.^® Plasmid DNA Mini Kit I (Omega Bio-tek, Inc., USA). Oligonucleotide synthesis and DNA sequencing were performed by Eton Bioscience, Inc. (USA). Genes were synthesized by GenScript, Inc. (USA). All strains used in this study are listed in Supporting Information Table S1.

Xue D., Shang Z., Older E.A., Zhong Z., Pulliam C., Peter K., Nagarkatti M., Nagarkatti P., Li Y.X, & Li J. (2023). Refactoring and Heterologous Expression of Class III Lanthipeptide Biosynthetic Gene Clusters Lead to the Discovery of N,N-Dimethylated Lantibiotics from Firmicutes. ACS chemical biology, 18(3), 508-517.

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Constructing Engineered Redox Sensor Strains

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Each single NADH dehydrogenase knockout and the ΔnuoNΔnqrF1 double-knockout strain were generated in a previous study and confirmed by complementation (Duhl et al., 2018 ). The Rex redox sensor plasmids were received from Dr. Vatsan Raman and Yang Liu at the University of Wisconsin—Madison) and were described in their recent publication (Liu et al., 2019 (link)). The two-plasmid system has one plasmid that constitutively expresses Rex and another that contains a fluorescent reporter under Rex regulation. The Rex-containing plasmid was transformed into chemically competent E. coli WM3064 cells. The plasmid was then transferred to S. oneidensis MR-1 and NADH dehydrogenase knockout strains via a conjugation protocol similar to Webster et al. (2014) (link). The plasmid containing the fluorescent reporter was transformed into chemically competent E. coli Mach 1 cells via heat shock. The Mach 1 cells were grown at 37°C while shaking for 18 hours in 5 mL cultures. The cultures were used to extract the reporter plasmid via the E.Z.N.A Plasmid DNA Mini Kit I (Omega Bio-tek, D6943–02). Extracted plasmid was then used to transform electrocompetent S. oneidensis MR-1 and NADH dehydrogenase strains containing the Rex plasmid via electroporation (Myers and Myers, 1997 (link)). The presence of both plasmids in each strain were confirmed with antibiotic resistance and PCR.

Duhl K.L, & TerAvest M.A. (2019). Shewanella oneidensis NADH dehydrogenase mutants exhibit an amino acid synthesis defect. Frontiers in energy research, 7, 116.

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Plasmid Extraction and Analysis

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The plasmids from Ab242 or from transformed A. nosocomialis cells were extracted using the Wizard® DNA purification kit (Promega, Madison, WI, USA) or the E.Z.N.A® Plasmid DNA Mini Kit I (OMEGA bio-tek, Norcross, GA, USA). The plasmids were analyzed by 0.7% agarose gel electrophoresis and ethidium bromide staining following conventional procedures (Sambrook et al., 1989 ).

Cameranesi M.M., Morán-Barrio J., Limansky A.S., Repizo G.D, & Viale A.M. (2018). Site-Specific Recombination at XerC/D Sites Mediates the Formation and Resolution of Plasmid Co-integrates Carrying a blaOXA-58- and TnaphA6-Resistance Module in Acinetobacter baumannii. Frontiers in Microbiology, 9, 66.

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Bacterial DNA Extraction and Amplification

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Genomic DNA was extracted using the TIANamp bacteria DNA kit (Spin Column) (Tiangen Biotech (Beijing) Co, Ltd., Beijing, China) following the protocol for isolating genomic DNA from bacteria. Bacterial plasmid DNA was isolated using the EZNA^® Plasmid DNA Mini Kit I (Omega Bio-tek Inc., Norcross, GA, USA). The concentration and purity of the DNA was measured using the Nano Drop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). All conventional PCR reactions were carried out in a Bioer XP Thermal Cycler (Hangzhou Bioer Tech. Co., Ltd., Hangzhou, China). Amplification of the DNA was performed in 50 µL total volumes with 2× TSINGKE Master Mix (TsingKe Biological Technology, Beijing, China) while the PCR conditions were 94 °C for 10 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 57~62 °C (specific for each gene) for 30 s, and extension at 72 °C for 1 min/kb.

Masum M.M., Yang Y., Li B., Olaitan O.S., Chen J., Zhang Y., Fang Y., Qiu W., Wang Y, & Sun G. (2017). Role of the Genes of Type VI Secretion System in Virulence of Rice Bacterial Brown Stripe Pathogen Acidovorax avenae subsp. avenae Strain RS-2. International Journal of Molecular Sciences, 18(10), 2024.

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