T vector pmd20

Manufactured by Takara Bio

Sourced in Japan, China, United States, Germany

The T-Vector pMD20 is a cloning vector provided by Takara Bio for the direct cloning of PCR products. It features a linear DNA structure with overhanging 3' dT ends, allowing for the seamless ligation of PCR products with 3' A-overhangs.

Automatically generated - may contain errors

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Close spelling variants of this product

T vector (47 citations) PMD20-T (16 citations) PMD20 vector (12 citations) PMD20 (9 citations) Takara T-Vector pMD20 (2 citations)

111 protocols using t vector pmd20

Characterization of BoTFL1-like Gene Function

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The open reading frame (ORF) of the BoTFL1-like cDNA was amplified using Pfu DNA Polymerase (BoCai Biotechnology, Shanghai, China) with forward primer ORFF and reverse primer ORFR, and inserted into a pMD20-T vector (Takara). Both pMD20-T vector and pCAMBIA1301 vector were digested with KpnI and XbaI (Takara) and then the BoTFL1-like ORF was cloned into the pCAMBIA1301 vector that contains the cauliflower mosaic virus (CaMV) 35S promoter. The resulting recombinant plasmid was then transformed into Agrobacterium tumefaciens strain GV3101 by electroporation using the Gene Pulser MXcell electroporation system (Bio-Rad, USA).
Arabidopsis plant transformation was carried out using the floral dipping method (Clough and Bent, 1998) . Transgenic plants were screened on 1/2 MS agar medium supplemented with hygromycin B (50 mg/L) before they were transplanted into soil. The flowering time and rosette leaf numbers of three T3 transgenic lines were counted when the first flower started to open on the main inflorescence, and floral structure was also screened. Each line had 30 repetitions. Insertion of the BoTFL1-like gene into the Arabidopsis genome was confirmed by PCR analysis using the primers ORFF and ORFR (Table 1).

Zeng H.Y., Lu Y.T., Yang X.M., Xu Y.H, & Lin X.C. (2015). Ectopic expression of the BoTFL1-like gene of Bambusa oldhamii delays blossoming in Arabidopsis thaliana and rescues the tfl1 mutant phenotype. Genetics and molecular research : GMR, 14(3).

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Constructing miRNA Expression Plasmids

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In order to construct the plasmids expressing the 43 miRNAs shown Supplementary Table 1, the C57BL/6 mouse genome was amplified with Ex Taq (TaKaRa Bio Inc., Kusatsu, Shiga, Japan) and primers (Supplementary Table 1), and the amplified DNA was ligated to a T-vector pMD20 (#3270; TaKaRa Bio Inc). Primers were designed with reference to mice chromosome region (Supplementary Table 1). DNA fragments were subcloned from the T-vector pMD20 into the restriction enzymes’ (Supplementary Table 1) site of the pmR-mCherry vector (#Z2542N; TaKaRa Bio Inc).

Ueno K., Kurazumi H., Suzuki R., Yanagihara M., Mizoguchi T., Harada T., Morikage N, & Hamano K. (2024). miR-709 exerts an angiogenic effect through a FGF2 upregulation induced by a GSK3B downregulation. Scientific Reports, 14, 11372.

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Isolation and Sequencing of Rhodopsin cDNA from Cryptomonas rhodomelas

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Total RNA was isolated from the C. rhodomelas eyeball using TRIzol Reagent (Life Technologies), and first-strand cDNAs were synthesized with SuperScript III reverse transcriptase (Life Technologies) using oligo(dT)₂₁ primer or KSII(dT)₂₁ (5’- GAGGT CGACG GTATC GATAA GCTTT TTTTT TTTTT TTTTT TTT -3’) primer. The CrRh cDNA fragments were amplified using Taq polymerase (Roche) and a pair of degenerate primers (df_rhoF1, 5’- TAYCC HCAGT AYTAC CTKG -3’ and df_rhoR2, 5’- GAACT GYYTG TTCAW SMARA TGTAG -3’) that were designed based on a conserved region of rhodopsin genes from the species Oryzias latipes, Takifugu rubripes, Xenopus tropicalis, Gallus gallus, and Bos taurus. Complementary DNAs including 5’- and 3’- UTRs were obtained using RACE. A pair of PCR primers (CrRh-full-F, 5’- GTCCG TCTCC ATCAC TCTCC GGAAG -3’ and CrRh-full-R, 5’- CTGTA GACGT TAGCC TTCAG TCGTT TC -3’) was designed in 5’- and 3’- UTRs to amplify cDNA fragments covering entire coding sequences with PfuUltra (Stratagene). The cDNA fragments were inserted into the pMD20-T vector (TaKaRa), and at least five independent clones were sequenced to obtain a full-length cDNA clone without presumed PCR errors.

Sakata R., Kabutomori R., Okano K., Mitsui H., Takemura A., Miwa T., Yamamoto H, & Okano T. (2015). Rhodopsin in the Dark Hot Sea: Molecular Analysis of Rhodopsin in a Snailfish, Careproctus rhodomelas, Living near the Deep-Sea Hydrothermal Vent. PLoS ONE, 10(8), e0135888.

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Determining Full-Length Transcripts via 5' RACE

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5′ rapid amplification of cDNA ends (RACE) was performed with 5′-Full RACE Core Set (Takara) to determine the full length sequences of Cs68435|c0_g1_i1 and Cs59318|c0_g1_i1. Total RNA was extracted from radular tissue as described above. 5′-RACE products were cloned into pMD20-T vector (Takara) and sequenced.

Nemoto M., Ren D., Herrera S., Pan S., Tamura T., Inagaki K, & Kisailus D. (2019). Integrated transcriptomic and proteomic analyses of a molecular mechanism of radular teeth biomineralization in Cryptochiton stelleri. Scientific Reports, 9, 856.

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Cloning and Transformation of DnAGL19

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The complete DnAGL19 open reading frame (KU373056) (Liang et al., 2012 (link)) was amplified from D. nobile cDNA that is reversely transcribed from total RNA extracted from axillary buds, followed by cloning into the pMD20-T vector (TaKaRa, Dalian, China) to sequence (Invitrogen, Shanghai, China). Primers for the full-length ORF amplification are listed in Supplementary Table S1. The DnAGL19 coding sequence (636-bp, with the stop codon removed) was inserted into the pCanG-Myc vector to generate the CaMV 35S::DnAGL19-6Myc construct, in which a sequence encoding six tandem repeats of a Myc-tag was fused to the 3′-end of the DnAGL19 ORF. The 35S::DnAGL19-6Myc construct was transformed into A. thaliana seedlings using the floral-dip method (Clough and Bent, 1998 (link)), and homozygote lines were selected for further analysis.

Liu X.R., Pan T., Liang W.Q., Gao L., Wang X.J., Li H.Q, & Liang S. (2016). Overexpression of an Orchid (Dendrobium nobile) SOC1/TM3-Like Ortholog, DnAGL19, in Arabidopsis Regulates HOS1-FT Expression. Frontiers in Plant Science, 7, 99.

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ADV PCR Mutation Analysis

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Purified ADV PCR products (400 ng) amplified from the genomic DNA extraction were re-annealed and treated with SURVEYOR nuclease (Transgenomics, Omaha, NE, USA) according to the manufacturer's recommended protocol. The products were analyzed on 10% TBE polyacrylamide gels, which were stained with SYBR Gold DNA stain (Life Technologies) and imaged using a Bio-Rad Gel Doc gel imaging system (Richmond, CA, USA). Quantification was based on the relative band intensities, as described by Cong et al. (2013) [12] (link). The PCR products were ligated into the pMD20-T vector (Takara Bio Inc.) and submitted for sequencing using universal primers (BGI, Guangzhou, China).

Bi Y., Sun L., Gao D., Ding C., Li Z., Li Y., Cun W, & Li Q. (2014). High-Efficiency Targeted Editing of Large Viral Genomes by RNA-Guided Nucleases. PLoS Pathogens, 10(5), e1004090.

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Engineered HTT Constructs for Genome Editing

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Constructs of E3a-, E3b-, E4-, and E6-GFP were generated based on the pcDNA3.1(+) vector using Gibson Assembly Master Mix (New England Biolabs, MA, USA). Specifically, different lengths of HTT fragments were amplified from a human cDNA library with 21 CAGs located in N-terminus. E3a∆-, E3b∆-, E4∆-, and E6∆-GFP plasmids were then, respectively, constructed by PCR using primers flanking exon 2 and 5′UTR from their corresponding plasmids and finally self-ligated into circle plasmids. The length of 5′UTR in all constructs is 145 bp, which is exactly the full-length of 5′UTR in the NM_002111 transcript. All pcDNA constructs were finally confirmed by Sanger sequencing. For genome editing, lentiCRISPRv2 (Addgene #52961) was used as the backbone, and sgHTT sequences were designed with the help of CHOPCHOP website (http://chopchop.cbu.uib.no/). In brief, sgRNA oligos were annealed and inserted into the BsmBI digested vector. Junctions of exon 1/2 or targeted genomic regions were amplified by PCR and ligated into the pMD20T vector (Takara Bio Inc., CA, USA) for T-A cloning. White colonies were picked out for plasmid extraction and Sanger sequencing. Sequences of sgHTT and PCR primers are listed in Table 1.

Wu J., Tang Y, & Zhang C.L. (2019). Targeting N-Terminal Huntingtin with a Dual-sgRNA Strategy by CRISPR/Cas9. BioMed Research International, 2019, 1039623.

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Recombinant Expression of IFN-CSP Protein

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The synthetic IFN-CSP gene fragments were cloned into the pMD20-T vector (Takara) and transformed into E. coli DH5a according to the procedures described by the manufacturer. The generated recombinant plasmids IFN-CSP/pMD20-T were digested and the inserts were cloned into Nde I/Xho I restriction sites of the expression vector pET-21b (Fig. 1a). The resulting expression plasmid IFN-CSP/pET-21b was finally transformed into E. coli BL21 (DE3) for IFN-CSP expression.Fig. 1

Schematic diagram of IFN-CSP gene in the expression vector IFN-CSP/pET-21b and expression of IFN-CSP protein in E. coli BL21/pET-21b-IFN-CSP. a: A Schematic diagram of IFN-CSP/pET-21b (T7 pro, T7 promoter; T7 ter, T7 terminator). b: SDS-PAGE analysis of protein expression. Lane M: Protein molecular weight marker, Lane 1–2: Total proteins of E. coli BL21/pET-21b-IFN-CSP before and after induction, Lane 3–4: Supernatant and precipitation after ultrasonication and centrifugation

Lu X., Wang J., Jin X, & Zhu J. (2015). High-level expression of a novel liver-targeting fusion interferon with preferred Escherichia coli codon preference and its anti-hepatitis B virus activity in vivo. BMC Biotechnology, 15, 54.

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Transient and Stable Overexpression of CEBPB/CEBPD in HK-2 Cells

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For transient overexpression studies, cDNA encoding human CEBPB or CEBPD was amplified from HK-2 cells using the primers listed in Table 1 and subcloned into the pMD20-T vector (Takara Bio). Each fragment was then subcloned into pcDNA3.1 (−) (Promega). For CEBPB/CEBPD double transfections, each vector amount was reduced to half the dose of single vector transfection (5 μg each, making total 10 μg vector/dish). HK-2 clones, which stably overexpress CEBPD, were generated through retrovirus transduction (Platinum Retrovirus Expression System, Pantropic, Cell Biolabs, San Diego, CA). The above CEBPD in the pMD20-T vector was further subcloned into the retroviral vector, pMXs-IRES-Puro, using the XhoI-SnaBI site. The plasmids were transfected to the packaging cell line, and, the culture supernatants were collected 24 and 48 h later, passed through 0.22-μm filters, and added to the cells. Drug selection by 4 μg/ml puromycin was initiated 72 h after infection.

Yamaguchi J., Tanaka T., Eto N, & Nangaku M. (2015). Inflammation and hypoxia linked to renal injury by CCAAT/enhancer-binding protein δ. Kidney International, 88(2), 262-275.

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Cold-adapted Cellulase EglC Cloning

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C. farmeri A1 that secreted cold-adapted EglC was isolated from R. labralis and stored in our laboratory. The plasmid pMD20-T vector (TaKaRa, Dalian, China) and E. coli DH5α were used for gene cloning, whereas the pET22b vector (Novagen, Madison, WI) and E. coli BL21 (laboratory stock) were used for gene expression. The flanking regions of the EglC gene were amplified using the TaKaRa Genome Walking Kit (TaKaRa, Dalian, China). Restriction endonucleases, T4 DNA ligase, and DNA polymerase were purchased from TaKaRa (Dalian, China). TIANgel Midi Purification Kit, TIANprep Mini Plasmid Kit, and 2 × Taq PCR Master Mix were purchased from Tiangen (Beijing, China).

Bai X., Yuan X., Wen A., Li J., Bai Y, & Shao T. (2016). Cloning, expression and characterization of a cold-adapted endo-1, 4-β-glucanase from Citrobacter farmeri A1, a symbiotic bacterium of Reticulitermes labralis. PeerJ, 4, e2679.

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