Plasmid purification kit

Manufactured by Tiangen Biotech

Sourced in China

The Plasmid Purification Kit is a laboratory equipment designed to isolate and purify plasmid DNA from bacterial cultures. It utilizes a simple and efficient protocol to extract and concentrate plasmid DNA, which can be used for various downstream applications such as cloning, sequencing, and transfection.

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Lab products found in correlation

Exosome free fbs, by System Biosciences (2 mentions) Agarose gel purification kit, by Qiagen (1 mentions) Pt18mobsacb, by Addgene (1 mentions) Bacterial genomic dna extraction kit, by Tiangen Biotech (1 mentions) Dh5a competent cells, by Tiangen Biotech (1 mentions) Pmd19 t, by Takara Bio (1 mentions) Peasy t1 vector, by Transgene (1 mentions) Trans tagtm high fidelitydna polymerase, by Transgene (1 mentions) Fitc phalloidin, by Merck Group (1 mentions) Total akt, by Proteintech (1 mentions) Fetal bovine serum (fbs), by Transgene (1 mentions) Wga fitc, by Merck Group (1 mentions) Exosome isolation reagent, by RiboBio (1 mentions) Dulbecco modified eagle medium (dmem), by Transgene (1 mentions) Ang 2, by Merck Group (1 mentions) β actin, by Proteintech (1 mentions) Sirnas, by RiboBio (1 mentions) P akt, by Proteintech (1 mentions) Restriction enzyme, by Takara Bio (1 mentions) Dntps, by Takara Bio (1 mentions)

Spelling variants of this product

Endotoxin-free plasmid purification kit (5 citations) Endo Free plasmid purification kit (4 citations) DNA purification and plasmid isolation kits (2 citations) Mini plasmid purification kit (2 citations)

7 protocols using plasmid purification kit

Genetic Manipulation of Mycobacterium neoaurum

Cited 1 time

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Mycobacterium neoaurum HGMS2, a non-pathogenic industrial Mycobacterium, was maintained in our laboratory and deposited at China Center for Type Culture Collection (CCTCC No: M2012522) [55 (link)] and its genome sequence was deposited in GenBank (CP031414.1). The plasmids p2NIL (Cat. 20188) [56 (link)] and pT18mobsacB (Cat. 72648) were purchased from AddGene (Watertown, MA, USA).
The genomic DNAs of Mycobacterium sp. HGMS2 and its mutants were extracted using a Bacterial Genomic DNA Extraction Kit from Tiangen (Beijing, China). Plasmids were purified using a Plasmid Purification Kit from Tiangen (Beijing, China). PCR fragments were purified using an agarose gel purification kit (Qiagen, USA).

Li X., Chen T., Peng F., Song S., Yu J., Sidoine D.N., Cheng X., Huang Y., He Y, & Su Z. (2021). Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria. Microbial Cell Factories, 20, 158.

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Quantification of PRRSV RNA via RT-qPCR

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To attain a relative quantity of viral RNA, TaqMan fluorescent quantitative RT-PCR (RT-qPCR) was performed on all serum samples as described previously^{40 (link)}. The PCR products of conserved regions within ORF7 for type 2 PRRSV strains (180 base pair) was cloned with the PMD-19T (Takara, Korea) and transformed into DH5a competent cells (TIANGEN, China). Plasmid DNA was extracted by using a plasmid purification kit (TIANGEN, China) and quantified by the Thermo Scientific Varioskan Flash multimode reader. Real-time RT-PCR using Taqman probes was performed to generate a standard curve by known amounts of the serially diluted ORF7-based plasmid standards (10¹–10⁸ copies/μL). Specific primers for qPCR in this study was performed as described^{40 (link)}, PRRSV F: 5′-ACAACGGCAAGCAGCAGAA-3′ and PRRSV R: 5′-GAGCGATGATCTTACCCAGCAT-3′ and the PRRSV probe: 5′-FAM-CTGGGYARGATYATCGCCCAGCA-BHQ1-3′. The concentrations in the tested samples were obtained by the Ct values plotted against the known concentration of the ORF7-based plasmid standards.

Wei C., Dai A., Fan J., Li Y., Chen A., Zhou X., Luo M., Yang X, & Liu J. (2019). Efficacy of Type 2 PRRSV vaccine against challenge with the Chinese lineage 1 (NADC30-like) PRRSVs in pigs. Scientific Reports, 9, 10781.

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Cloning and Expression of T. gondii SAG4

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The entire coding sequences of the T. gondii SAG4 gene were amplified by PCR from genomic DNA of T. gondii strain RH with synthetic primers. Trans Tag^TM High FidelityDNA Polymerase (TransGen, Beijing, China) was used in PCR amplification.
SAG4: Forward primer: 5′-GGGGTACCATGACGAAAAATAAAATT-3′ Reverse primer: 5′-CGGGATCCTTACATTGATATCAACA-3′ (introduced KpnI and BamH I recognition sites, respectively, are underlined).
The PCR production of SAG4 gene was cloned into the pEASY-T1 Vector (TransGen, Beijing, China), digested with the appropriate restriction enzyme (KpnI and BamH I), and purified from agarose gels. SAG4 gene fragment was inserted into the eukaryocyte vector pEGFP-C1, generating pSAG4. The recombinant plasmids were then propagated in Escherichia coli DH5α and confirmed by restriction analysis and sequencing. Endotoxin-free plasmid DNA was isolated using a Plasmid Purification Kit (TianGen, Beijing, China). The concentrations of the purified plasmids were detected by spectrophotometer at 260 and 280 nm, and the 260:280 ultraviolet absorption ratio was between 1.8 and 2.0. All the plasmids were diluted into 1 mg/ml by sterile endotoxin-free PBS and stored at -20°C before use.

Zhou J, & Wang L. (2017). SAG4 DNA and Peptide Vaccination Provides Partial Protection against T. gondii Infection in BALB/c Mice. Frontiers in Microbiology, 8, 1733.

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Codon-Optimized Ex160-cDNA Expression

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The Ex160-cDNA was synthesized with codon optimisation (S1 Data) for procaryotic expression (GenScript Biotech Co., Ltd., Nanjing, China) and cloned into BamHI/HindIII restriction sites in the same reading frame with the His tag in the pQE-30 vector. The recombinant plasmid was transformed into E. Coli DH5α. The plasmid was extracted from the bacteria using a plasmid purification kit (Tiangen Biotech Co., Ltd., Beijing, China), and digested by BamHI/HindIII enzymes (NEB, Beverly, MA, USA) to confirm ligation of this gene into the pQE-30 plasmid. Agarose gel electrophoresis was performed to confirm the correct insertion of the target gene.

Zhu J., Zhang L., Xue Z., Li Z., Wang C., Chen F., Li Y., Dai Y., Zhou Y., Zhou S., Chen X., Okumura-Noji K., Lu R., Yokoyama S, & Su C. (2023). Vaccination against the HDL receptor of S. japonicum inhibits egg embryonation and prevents fatal hepatic complication in rabbit model. PLoS neglected tropical diseases, 17(11).

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RT-LAMP Assay for SARS-CoV-2 Delta Variant

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We tracked the SARS-CoV-2 genome sequences from the GISAID database and analyzed real-time mutation situation reports from the standardized, open-source database of COVID-19 (https://outbreak.info/). Target mutation sites were selected from typical mutations in the Delta variant, and the sequence region containing the mutation was analyzed by BLASTN to ensure that the region used for designing the RT-LAMP primers was specific to Delta. In addition, the conservative fragments that did not contain any mutations in the N gene were selected as the “internal reference” template.
The wild-type SARS-CoV-2 N gene plasmid (GenBank MN908947.3, 28274∼29533nt) was synthesized by Tsingke Biotechnology. The synthetic wild-type N gene plasmid was used as a wild-type positive control in the RT-LAMP reaction. Site-directed mutagenesis was performed on the 608th nucleotide of the wild-type N gene plasmid, which is the R203M mutation (G→T). Similarly, R203K/G204R (GGG→AAC) and T205I (C→T) mutations were also prepared. Then, the mutant plasmid was purified with a plasmid purification kit (TIANGEN BIOTECH), and sequenced by Tsingke Biotechnology. The quality and concentration of DNA were determined with a Nanodrop UV-Vis spectrophotometer, and the nucleotide copy number was calculated accordingly.

Yang J., Hu X., Wang W., Yang Y., Zhang X., Fang W., Zhang L., Li S, & Gu B. (2022). RT-LAMP assay for rapid detection of the R203M mutation in SARS-CoV-2 Delta variant. Emerging Microbes & Infections, 11(1), 978-987.

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Exosome Isolation and Analysis

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DMEM and fetal bovine serum (FBS) were obtained from Transgen Biotech (Beijing, China). Exosome-free FBS was from SBI (San Francisco, CA). Ang II, FITC-phalloidin, and FITC-WGA were from Sigma-Aldrich (St. Louis, MO). Antibodies against BNP and ANP were purchased from Abcam (Cambridge, MA), whereas PTEN, p-AKT, total AKT, β-actin, and CD63 were from Proteintech (Chicago, IL). Plasmid purification kits were from TIANGEN (Beijing, China). Chemically synthesized miRNAs, siRNAs, and exosome isolation reagent were obtained from RiboBio (Guangzhou, China).

Nie X., Fan J., Li H., Yin Z., Zhao Y., Dai B., Dong N., Chen C, & Wang D.W. (2018). miR-217 Promotes Cardiac Hypertrophy and Dysfunction by Targeting PTEN. Molecular Therapy. Nucleic Acids, 12, 254-266.

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Genetic Modification of Mycobacterium neoaurum

Cited 2 times

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Mycobacteriumneoaurum HGMS2 was maintained in our laboratory, and its genome sequence is available in GenBank (CP031414.1) [51 (link)]. The homology recombination vector p2NIL-Sac was constructed previously [52 (link)]. The plasmid pMV 261 was purchased from AddGene (Watertown, MA, USA). Restriction enzymes, dNTPs, and Taq and Pfu DNA polymerases were purchased from Takara Co. (Dalian, China). Other molecular biology reagents were of the highest grade and were obtained from New England Biotech (MA, USA). Genomic DNA extraction kits, plasmid purification kits and PCR purification kits were obtained from Tiangen (Beijing, China).
Standard samples of 4-androstene-3,17-dione (4-AD), 22-hydroxy-23,24-bisnorchol-1,4-diene-3-one (DBA), 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one (9OH-BA), and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) were prepared by Amersino (Wuhan, China). Phytosterol (98%, 410.40 Da) [52 (link)] were obtained from Hubei Goto Pharmaceutical Co. (Xiangyang, China).

Song S., He J., Gao M., Huang Y., Cheng X, & Su Z. (2023). Loop pathways are responsible for tuning the accumulation of C19- and C22-sterol intermediates in the mycobacterial phytosterol degradation pathway. Microbial Cell Factories, 22, 19.

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