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Miniprep kit

Manufactured by Macherey-Nagel
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The Miniprep kit is a laboratory tool designed for the rapid and efficient purification of plasmid DNA from bacterial cultures. It provides a simple and reliable method for extracting small amounts of plasmid DNA, which can be used for various applications, such as sequencing, cloning, and transfection.

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Lab products found in correlation

Pgem t easy vector, by Promega (1 mentions) Nucleospin gel and pcr clean up, by Macherey-Nagel (1 mentions) Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions) Rapid dna ligation kit, by Thermo Fisher Scientific (1 mentions) Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions) E coli top10 cells, by Thermo Fisher Scientific (1 mentions) Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions) Muller hinton 2 agar, by BD (1 mentions) Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions) Esf 921 medium, by Expression Systems (1 mentions) Rnaimax, by Thermo Fisher Scientific (1 mentions) Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions) Fumaric acid, by Merck Group (1 mentions) Succinic acid, by Merck Group (1 mentions) Ni sepharose 6 fast flow, by GE Healthcare (1 mentions) Prepacked pd 10 sephadex g 25 columns, by GE Healthcare (1 mentions) T4 dna ligase, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

NucleoSpin plasmid miniprep kit (27 citations) NucleoSpin RNA miniprep kit (6 citations) NucleoSpin RNA Plus miniprep kit (4 citations) 96well miniprep kit (3 citations) Plasmid Miniprep Kit (3 citations) NucleoSpin 96-well Miniprep kits (2 citations) NucleoBond PC 100 Plasmid Miniprep Kit (1 citations)

10 protocols using miniprep kit

1

qPCR Primer Design and Validation

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All primers for qPCR were designed using BatchPrimer3 v1.0 based on the target genes annotated in the latest cod genome assembly gadMor2 (Table S8) (14 (link)). Target genes were validated using thermal gradient RT-PCR and the products that met the quality criteria were cloned into bacterial plasmids. Two micrograms of cDNA were used as a template for PCR with gene-specific primers. Target mRNAs were amplified using MyTaq HS DNA Polymerases (Bioline, UK), and amplicons were run on 1% agarose gels, stained with ethidium bromide and purified with NucleoSpin® Gel and PCR Clean-up (Macherey-Nagel, Germany). Purified PCR products were ligated in pGEM-T easy vectors (Promega, USA) and transformed into Escherichia coli (DH5a strain). One selected transformant of each construct was grown to obtain plasmid DNA (Miniprep kit, Macherey-Nagel). All constructs were verified by Sanger DNA sequencing with T7 and SP6 primer sets (GATC Biotech, Table S8). Pro-inflammatory cytokines mRNAs for il1b and il6 were selected on account of their high inducibility during PAMP responses (Figure S7). See details for PCR program and reaction systems in supplementary Information. One-way ANOVA was used to test the statistical differences between indicated experimental contrast. Significance was reported if P < 0.05. Graphs were plotted using Prism6® (Table S5).
Jin X., Morro B., Tørresen O.K., Moiche V., Solbakken M.H., Jakobsen K.S., Jentoft S, & MacKenzie S. (2020). Innovation in Nucleotide-Binding Oligomerization-Like Receptor and Toll-Like Receptor Sensing Drives the Major Histocompatibility Complex-II Free Atlantic Cod Immune System. Frontiers in Immunology, 11, 609456.
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2

Relative Plasmid Copy Number Quantification

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Relative plasmid copy numbers were determined based upon fluorescence intensity of agarose gel bands corresponding to linearized plasmid DNA harvested from identical numbers of cells. Three colonies were picked from a fresh transformation and grown to saturation in LB broth containing 100 μg/ml of carbenicillin. The cultures were normalized according to OD600 absorbance in order to start with constant dry weight of cells. Plasmids were prepped from these normalized suspensions using a plasmid miniprep kit (Machery-Nagel, cat. #740499.250) and 10 μl of prep were linearized with the DpnI enzyme, which has a single recognition site on the pGFPuv plasmid. Linear fragments were separated in a 1% agarose gel, stained with Ethidium Bromide, and visualized using a UVP BioSpectrum 300 LM26E transilluminator equipped with a EtBr filter (570–640 nm). The shutter speed was set to 126 ms. Camera aperture and image contrast were adjusted so that none of the fluorescent bands saturated the image. Band intensity was measured by densitometry using Image J software, with fixed area size for all the samples. The results were expressed as fold increase relative to background.
Standley M., Million-Weaver S., Alexander D., Hu S, & Camps M. (2018). Genetic control of ColE1 plasmid stability that is independent of plasmid copy number regulation. Current genetics, 65(1), 179-192.
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3

Site-directed Mutagenesis of ϕX174 dsDNA

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The ϕX174 dsDNA replicative form was purified from infected cultures before lysis using a standard miniprep kit (Macherey-Nagel), and 500 pg of this DNA were used as template for polymerase chain reaction-based mutagenesis using Phusion high-fidelity DNA polymerase (Thermo Scientific) and contiguous, divergent, 5’-phosphorylated primers, of which the reverse primer carried the desired nucleotide substitution. Polymerase chain reaction products were circularized with the Rapid DNA ligation kit (Thermo Scientific) and used for transfecting competent IJ1862cells by the classical heat-shock method. A single plaque was picked, resuspended Lysogeny Broth medium, and stored at –70°C. The presence of each substitution was confirmed by Sanger sequencing. This process was iterated until twenty GATC sites were introduced. Full-length sequencing of the 20GATC virus was performed to verify that all mutations were present and that no other changes were introduced.
Pereira-Gómez M, & Sanjuán R. (2015). Effect of mismatch repair on the mutation rate of bacteriophage ϕX174. Virus Evolution, 1(1), vev010.
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4

Protein Expression Profiling via NAPPA Arrays

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NAPPA arrays were manufactured as previously described22 (link),23 (link). Briefly, bacterial clones having the genes of interest with a GST tag at the c-terminus, were obtained from the DNASU Plasmid Repository (DNASU.org). Plasmid DNA was purified using a mini-prep kit (Macherey–Nagel, #740499.50). DNA concentrations were then measured and normalized to 100 ng/µl for all 1700 genes. Silicon nanowell substrates were coated with (3-Aminopropyl) triethoxysilane (APTS) (Thermo Scientific, #80370) and then the plasmid DNA was printed using a piezo electric printer. At the time of usage, proteins were expressed from plasmid DNA using an in-vitro transcription and translation (IVTT) kit (Thermo Scientific, #88882). The printing quality of a batch was determined by expressing a random slide from the batch with the IVTT kit, followed by the detection of GST-tagged proteins with Mouse anti-GST antibody (Cell Signaling, #2624S) and Alexa 555 Goat anti-mouse IgG antibody (Invitrogen, #A-21422).
O’Kell A.L., Shome M., Qiu J., Williams S., Chung Y., LaBaer J., Atkinson M.A, & Wasserfall C. (2022). Exploration of autoantibody responses in canine diabetes using protein arrays. Scientific Reports, 12, 2490.
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5

CRISPR Library Generation in E. coli

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The library was designed by randomly choosing targets with a proper NGG PAM around the genome of E. coli strain MG1655 (NC_000913.2). A pool of 92,919 oligonucleotides (synthesized by CustomArray) was amplified with primers LC296 and LC297 using the Phusion DNA polymerase (Thermo Scientific) over 18 cycles. The psgRNA backbone was PCR amplified using primers LC293 and LC294. Both the vector backbone and library insert were gel purified, followed by Gibson assembly. To avoid the introduction of bottlenecks in the library, a total of 19 transformation assays were performed each using 0.2 μl of Gibson assembly product and 20 μl of MG1655 electro-competent cells and plated on 12 × 12 cm2 petri dishes resulting in a total of about 107 colonies. Colonies were allowed to grow for only 4–5 h at 37 °C before pooling all the cells together. The plasmid library was then extracted from 5 ml of pooled colonies using a miniprep kit (Macherey-Nagel). The resulting plasmid library DNA was further transferred to strains LC-E18 and LC-E75 by electroporation.
Cui L., Vigouroux A., Rousset F., Varet H., Khanna V, & Bikard D. (2018). A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9. Nature Communications, 9, 1912.
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6

Generation of Barcoded Plasmid Library

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Barcoded plasmids were generated in (Cira et al., 2018 (link)). To generate the barcoded library in E. coli MG1655, plasmid DNA was extracted from TOP10 E. coli cells (ThermoFisher) with a miniprep kit according to the manufacturer’s instructions (Macherey-Nagel). Plasmids were transformed into chemically competent E. coli MG1655 cells via heat shock and plated on LB agar plates containing chloramphenicol (35 μg/mL) and ampicillin (100 μg/mL). Colonies were picked, grown separately in LB broth with chloramphenicol (35 μg/mL) and ampicillin (100 μg/mL), and banked as glycerol freezer stocks at −80 °C.
Vasquez K.S., Willis L., Cira N.J., Ng K.M., Pedro M.F., Aranda-Díaz A., Ranjendram M., Yu F.B., Higginbottom S.K., Neff N., Sherlock G., Xavier K.B., Quake S.R., Sonnenburg J.L., Good B.H, & Huang K.C. (2021). Quantifying rapid bacterial evolution and transmission within the mouse intestine. Cell host & microbe, 29(9), 1454-1468.e4.
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7

Site-Directed Mutagenesis of nrdJ Gene

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The introduction of mutations for targeted amino acid exchange was performed by Ω-PCR with the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies). As a template, the expression plasmids pET28b(+) were applied, containing the respective nrdJ gene between the restriction sites NdeI and HindIII (16 , 17 (link)). The mutagenesis was performed according to the manufacturer's instructions from the QuikChange II Site-Directed Mutagenesis Kit. The mutagenesis product was transformed into electrocompetent Escherichia coli TOP10. The identity of the variant was confirmed by plasmid preparation (Miniprep kit, Macherey-Nagel GmbH & Co. KG) and sequencing (Microsynth Seqlab GmbH). Plasmids with the confirmed mutation were transformed into E. coli BL21(DE3) for recombinant gene expression.
Schell E., Nouairia G., Steiner E., Weber N., Lundin D, & Loderer C. (2021). Structural determinants and distribution of phosphate specificity in ribonucleotide reductases. The Journal of Biological Chemistry, 297(2), 101008.
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8

Bacterial Strain Cultivation Protocol

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All of the strains and plasmids used in this study are detailed in Dataset S3. Experiments were performed using Muller Hinton II agar or cation adjusted broth (Becton Dickinson) unless specified. Antibiotics were supplied by Sigma-Aldrich and were used at the following concentrations: carbenicillin 100 µg/mL, chloramphenicol 30 µg/mL, nalidixic acid 30 µg/mL, streptomycin 100 µg/mL, rifampicin 100 µg/mL, trimethoprim 2 µg/mL and 16 µg/mL, and tetracycline 15 µg/mL. Cultures were routinely grown at 37 °C with shaking (225 rpm). Plasmids were extracted using a commercial miniprep kit (Macherey-Nagel) and were transformed into TSS competent cells (46 (link)).
Rodríguez-Beltrán J., Sørum V., Toll-Riera M., de la Vega C., Peña-Miller R, & San Millán Á. (2020). Genetic dominance governs the evolution and spread of mobile genetic elements in bacteria. Proceedings of the National Academy of Sciences of the United States of America, 117(27), 15755-15762.
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9

Bacterial Expression and Cell Culture Protocol

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All bacteria were cultured in Luria–Bertani medium with appropriate antibiotics at 37°C. Plasmids were maintained in E. coli XL-1 Blue (Stratagene, San Diego, CA) and purified using standard miniprep kits (Machery-Nagel, Bethlehem, PA) before transfection. During bacterial expression, GST-fusion proteins were produced in E. coli Rosetta(DE3)pLysS (EMD Millipore, Billerica, MA). Sf9 insect cells were grown in ESF921 medium (Expression Systems, Davis, CA) at 28°C and infected with baculoviruses encoding MBP- or GST-fusion proteins at a multiplicity of infection of ∼1. Cos7, HeLa, and HFF cells were cultured in DMEM plus 10% fetal bovine serum and antibiotic-antimycotic (Invitrogen) at 37°C in 5% CO2. Transfections with DNA or RNA used Lipofectamine-LTX or RNAiMAX (Invitrogen), respectively (Campellone et al., 2008 (link)). Control siRNAs, siRNAs to glyceraldehyde-3-phosphate dehydrogenase, and siRNA pair #3 to Rab1a+b were from Ambion, and siRNA pairs #1 and 2 to Rab1a+b and siRNAs #1 and 2 to RhoD were from Sigma-Aldrich (St. Louis, MO). For selection of HFF clones stably transfected with LAP or LAP-WHAMM, media were supplemented with 750 μg/ml G418. To induce LAP expression, 7.6 mM sodium butyrate was added for 12–18 h.
Russo A.J., Mathiowetz A.J., Hong S., Welch M.D, & Campellone K.G. (2016). Rab1 recruits WHAMM during membrane remodeling but limits actin nucleation. Molecular Biology of the Cell, 27(6), 967-978.
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10

Purification and Characterization of Bacterial Enzymes

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Ingredients for buffers and media were obtained
from Duchefa Biochemie (Haarlem, The Netherlands) or Merck (Darmstadt,
Germany). All other chemicals used in the experiments, including the
sodium salt of (S,S)-EDDS, fumaric
acid, and succinic acid, were purchased from Sigma-Aldrich Chemical
Co. (St. Louis, MO) unless stated otherwise. Molecular biology reagents,
including restriction enzymes, polymerase chain reaction (PCR) reagents,
T4 DNA ligase, and DNA and protein ladders, were obtained from Fermentas
(ThermoFisher Scientific, Pittsburgh, PA) or Promega Corp. (Madison,
WI). PCR purification, gel extraction, and Miniprep kits were provided
by Macherey-Nagel (Duren, Germany). Ni-Sepharose 6 Fast Flow and prepacked
PD-10 Sephadex G-25 columns were purchased from GE Healthcare Life
Sciences (Uppsala, Sweden). Primers for DNA amplification were synthesized
by Eurofins MWG Operon (Cologne, Germany).
Poddar H., de Villiers J., Zhang J., Puthan Veetil V., Raj H., Thunnissen A.M, & Poelarends G.J. (2018). Structural Basis for the Catalytic Mechanism of Ethylenediamine-N,N′-disuccinic Acid Lyase, a Carbon–Nitrogen Bond-Forming Enzyme with a Broad Substrate Scope. Biochemistry, 57(26), 3752-3763.
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