Nucleospin plasmid kit

Manufactured by Takara Bio

Sourced in Germany

The NucleoSpin Plasmid kit is a product for the purification of plasmid DNA from bacterial cultures. It facilitates the isolation of high-quality plasmid DNA suitable for downstream applications such as cloning, sequencing, and transfection.

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Lab products found in correlation

Primestar gxl dna polymerase, by Takara Bio (1 mentions) Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions) Pcdna3, by Addgene (1 mentions) Xl1 blue supercompetent cells, by Agilent Technologies (1 mentions) Chloroquine, by Merck Group (1 mentions) Plasmid midi kit, by Qiagen (1 mentions)

Spelling variants of this product

NucleoSpin kit (17 citations) NucleoSpin Plasmid miniprep kit (2 citations)

4 protocols using nucleospin plasmid kit

Efficient Gene Cloning and Sequencing

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81.

Synthesize cDNA from your isolated RNA using the QuantiTect Reverse Transcription kit and its enclosed random hexamers primer mix (see key resource table).

82.

Amplify the gene of interest from cDNA with Takara PrimeSTAR GXL DNA Polymerase.

83.

Ligate the PCR product into pJET1.2 following instructions of CloneJet PCR cloning kit (Figure 2A).

84.

Transform chemically competent TOP10 cells with the ligation product and plate on carbenicillin selection plates.

85.

Perform a colony PCR with a standard Taq polymerase on at least 5 colonies.

86.

Isolate plasmids containing the gene of interest with the NucleoSpin Plasmid kit.

87.

Sequence the pJET plasmids containing your gene of interest and continue with the protocol if results match expectations.

Zöllner N.R., Bezrutczyk M., Laureyns R., Nelissen H., Simon R, & Frommer W.B. (2021). An RNA in situ hybridization protocol optimized for monocot tissue. STAR Protocols, 2(2), 100398.

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Engineered SARS-CoV-2 Spike Protein Variants

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pLHs1–24 containing zDHHS enzymes in modified pEG BacMam vectors, and M1–M7, Δcys, and C1-C6 were generated using and QuikChange II Site-Directed Mutagenesis Kit (Agilent). M1-M7, Δcys, and C1-C6 DNA were cloned from pcDNA3.1 (Addgene) containing Human Codon-Optimized 2019-nCov-S Protein. All plasmids were transformed into XL1-Blue super-competent Cells (Agilent), subsequently miniprepped with NucleoSpin Plasmid kit (Takara Bio) and sequence verified.

Puthenveetil R., Lun C.M., Murphy R.E., Healy L.B., Vilmen G., Christenson E.T., Freed E.O, & Banerjee A. (2021). S-acylation of SARS-CoV-2 spike protein: Mechanistic dissection, in vitro reconstitution and role in viral infectivity. The Journal of Biological Chemistry, 297(4), 101112.

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Supercoiled Plasmid Visualization in E. coli BL21(DE3)

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The E. coli BL21(DE3) carrying pET-topAI was cultured in M9-glucose at 37°C. When O.D.₆₀₀ of the culture reached 0.6, different amounts of IPTG were added as indicated in Figure 3. A 20-ml culture was taken at indicated times and centrifuged with 5000 r.p.m. at 4°C for 10 min. The pET-topAI plasmid was extracted from the collected cells by using NucleoSpin Plasmid kit (Clontech) and 800 ng of plasmid DNA was analyzed by electrophoresis on 1.0% agarose gel containing 2.5 μg/ml chloroquine (Sigma) in Tris-borate EDTA (TBE) buffer. The gel was run for 12 h at 3 V/cm at room temperature, washed with water for 2 h and stained with ethidium bromide. The DNA bands were then visualized by UV trans-illumination.

Yamaguchi Y, & Inouye M. (2015). An endogenous protein inhibitor, YjhX (TopAI), for topoisomerase I from Escherichia coli. Nucleic Acids Research, 43(21), 10387-10396.

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Transfection of Jurkat T Cells with TCR Constructs

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Recombinant pMICherry plasmids with full-length TCRαβ or TCRγδ inserts were isolated in small scale by using a NucleoSpin Plasmid kit (Clontech, Mountain View, CA) and in large scale for transfection using a Plasmid Midi kit (Qiagen, Hilden, Germany) per the manufacturer’s instructions. The Neon Transfection System was used to transfect 10 μg TCRαβ or γδ DNA in the pMICherry vector into the human Jurkat 76 TCRα^–β^– cell line (2 × 10⁷ cells/ml, 100 μl),^{65 (link)} followed by three pulses with a voltage of 1,350V and a width of 10 ms. The transfected cells were cultured for 48 hours before being assayed for TCRαβ or TCRγδ expression on the surface by FACS analysis. The human Jurkat 76 cells TCRα^–β^– cell line was cultured in complete-RPMI 1640 medium, which is RPMI 1640 with 10% of fetal bovine serum, 1% Penicillin Streptomycin, and 1% L-glutamine at 37 °C and 5% CO₂.

Guo X.Z., Dash P., Calverley M., Tomchuck S., Dallas M.H, & Thomas P.G. (2016). Rapid cloning, expression, and functional characterization of paired αβ and γδ T-cell receptor chains from single-cell analysis. Molecular Therapy. Methods & Clinical Development, 3, 15054-.

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