Topo directional cloning

Manufactured by Thermo Fisher Scientific

TOPO directional cloning is a molecular biology technique used for the efficient insertion of DNA fragments into plasmid vectors. It utilizes the topoisomerase I enzyme from the Vaccinia virus to facilitate the direct ligation of PCR products into the vector, enabling a rapid and directional cloning process.

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Lab products found in correlation

Close spelling variants of this product

PENTR Directional TOPO Cloning Kit (61 citations) TOPO cloning (47 citations) PLenti6/V5 Directional TOPO Cloning Kit (10 citations) Directional TOPO cloning kit (6 citations) PENTR Directional TOPO Cloning vector (3 citations)

7 protocols using topo directional cloning

Constructing HA- and FLAG-tagged KLF15

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HA- and FLAG-tagged KLF15 were constructed by PCR using untagged human KLF15 purchased from the Arizona State University plasmid repository as a template using and following primers: HA-KLF15-Forward-5′-GGG CCC GGA TCC ATG TAC CCC TAC GAC GTG CCC GAC TAC GCC GTG GAC CAC TTA CTT -3′; HA-KLF15-Reverse-5′GGG CCC CTC GAG CTA GTT CAC GGA GCG CAC GG-3′- -3′. 135 amino acids at KLF15 C-terminus corresponding to zinc-finger domain in KLF15 were truncated using following primers: Forward:5′-GGG CCC GAA TTC CTA AAT GCG CAC AAA CTT GGA GGG C-3′; Reverse:5′-GGG CCC GAA TTC CTA GTT CAC GGA GCG CAC GG-3′.
The PCR product was cloned into pcDNA3.1 using directional topo cloning (Invitrogen) Adenoviruses encoding HA and FLAG tagged KLF15 was constructed by subcloning into entry PENTR1a vector using and restriction enzymes followed by LR recombination with destination vector PDEST. The HDAC2-PDEST DNA was digested with PacI, ethanol precipitated and transfected into 293 HEK cells. After cytopathic effect (CPE), adenoviruses were collected and purified via three freeze-thaw cycles and a Millipore adenovirus purification Kit.

Pandey D., Nomura Y., Rossberg M.C., Hori D., Bhatta A., Keceli G., Leucker T., Santhanam L., Shimoda L.A., Berkowitz D, & Romer L. (2018). Hypoxia Triggers SENP1 Modulation of Kruppel-Like Factor 15 and Transcriptional Regulation of Arginase 2 in Pulmonary Endothelium. Arteriosclerosis, thrombosis, and vascular biology, 38(4), 913-926.

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Overexpression of IMPA-1 Gene in Plants

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The coding sequence of the IMPA-1 gene or the impa-1^G146E allele was amplified from genomic templates using a high-fidelity thermostable DNA polymerase (Phusion; New England Biolabs) and primers IMPA-1_dTF1: 5′-CACCTACATTAGCTTGCCTCAAAGAACAC-3′ and IMPA1_ORF_R2: 5′-TCAGCTGAAGTTGAATCCTCCGGAT-3′. The amplified regions contained all exons (including the stop codon) and introns, the annotated 5′-UTR and an additional 167 base pairs upstream. These amplification products were inserted into Gateway entry vector (pENTR/D-TOPO) using directional TOPO cloning (Invitrogen/Life Technologies). Sequence-verified inserts were moved via LR Clonase reactions (Invitrogen/Life Technologies) into the Gateway destination vector pEG100 (Earley et al., 2006 (link)), which contains T-DNA borders for Agrobacterium-mediated plant transformation, a Cauliflower Mosaic Virus 35S promoter to amplify expression, and an OCS 3′ end. Validate constructs were then electroporated into Agrobacterium strain LBA4404, and crwn4-2 mutant plants were transformed using the floral dip method (Bent, 2006 (link)). Transformants were identified among the progeny by selection for resistance to the herbicide Basta (glufosinate).

Blunt E.L., Shandler J.A., Hughes E.J., Sussman H., Christopherson R.C, & Richards E.J. (2020). Coordination of NMCP1- and NMCP2-class proteins within the plant nucleoskeleton. Molecular Biology of the Cell, 31(26), 2948-2958.

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Cloning and Expression of PTEN in GBM

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The mRNA from each GBM cells was prepared using an RNeasy kit (Qiagen) according to manufacturer’s protocol. The PTEN cDNA was synthesized from mRNA using the following primer pair: 5’-ctgggatccaaataaaaATGACAGCCATCATCAAAGAGATCG-3’ and 5’-ctcctcgagccGACTTTTGTAATTTGTGTATGCTGATCTTC-3’ using the OneStep RT-PCR kit (Qiagen). It was then amplified using the same primer pair with Phusion DNA polymerase (Thermo Fisher Scientific). GAPDH was used as a control and was amplified using the primer pair 5’-ATGGGGAAGGTGAAGGTCGGA-3’ and 5’-TTACTCCTTGGAGGCCATGTGGG-3’. The PTEN cDNA from each GBM cell was cloned into a pCR-Blunt II-TOPO vector using a Zero Blunt TOPO PCR cloning kit (Life Technologies). GFP was fused to the 3’-end of PTEN using overlapping PCR. It was then cloned into the pcDNA3.1-TOPO vector using the primer pair 5’-caccATGACAGCCATCATCAAAG-3’ and 5’-TTACTTGTACAGCTCGTCCATG-3’ through directional TOPO cloning (Life Technologies).
The plasmids and PCR primers used in this study are listed in Table S1. PTEN variants were generated using overlapping extension PCR ⁵¹, and they were cloned into either the mammalian expression vector pcDNA3.1 (Life Technologies), peGFP-C1 (Clontech), or the lentiviral vector pHR-SIN (Nghia). All constructs were confirmed through DNA sequencing.

Yang J.M., Schiapparelli P., Nguyen H.N., Igarashi A., Zhang Q., Abbadi S., Amzel L.M., Sesaki H., Quiñones-Hinojosa A, & Iijima M. (2017). Characterization of PTEN mutations in brain cancer reveals that pten mono-ubiquitination promotes protein stability and nuclear localization. Oncogene, 36(26), 3673-3685.

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Cloning and Expression of PTEN in GBM Cells

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The mRNA from each GBM cells was prepared using an RNeasy kit (Qiagen, Hilden, Germany) according to manufacturer’s protocol. The PTEN cDNA was synthesized from mRNA using the following primer pair: 5′-ctgggatccaaataaaaATGACAGCCATCATCAAAGAGATCG-3′ and 5′-ctcctcgagccGACTTTTGTAATTTGTGTATGCTGATCTTC-3′ using the OneStep RT-PCR kit (Qiagen). It was then amplified using the same primer pair with Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA). GAPDH was used as a control and was amplified using the primer pair 5′-ATGGGGAAGGTGAAGGTCGGA-3′ and 5′-TTACTCCTTGGAGGCCATGTGGG-3′. The PTEN cDNA from each GBM cell was cloned into a pCR-Blunt II-TOPO vector using a Zero Blunt TOPO PCR cloning kit (Life Technologies, Waltham, MA, USA). GFP was fused to the 3′-end of PTEN using overlapping PCR. It was then cloned into the pcDNA3.1-TOPO vector using the primer pair 5′-caccATGACAGCCATCATCAAAG-3′ and 5′-TTACTTGTACAGCTCGTCCATG-3′ through directional TOPO cloning (Life Technologies).
The plasmids and PCR primers used in this study are listed in Supplementary Table S1. PTEN variants were generated using overlapping extension PCR,⁴¹ and they were cloned into either the mammalian expression vector pcDNA3.1 (Life Technologies), peGFP-C1 (Clontech, Mountain View, CA, USA), or the lentiviral vector pHR-SIN. All constructs were confirmed through DNA sequencing.

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Cloning and Expressing Zebrafish Mcm2 and Mcm7

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All PCRs for cloning of expression constructs were performed using Phusion polymerase (NEB). Full length zebrafish Mcm2 was amplified from 24 hours post fertilization (hpf) zebrafish cDNA and cloned by TOPO directional cloning (Invitrogen) into pcDNA3.1 with C-terminal His and V5 tags. To facilitate rescue constructs after antisense morpholino oligonucleotide (MO) injection the MO binding site was silently mutated. To generate the open reading frame (ORF) of zebrafish Mcm7, 5′-RACE PCRs (FirstChoice™ RLM-RACE Kit, Thermo Fisher) based on ENSDART00000159300.2 were performed on RNA from 13 somites stage (ss) embryos. The ORF (Genbank accession no. MH746781) was amplified from 24 hpf zebrafish cDNA and cloned by directional TOPO cloning as described for Mcm2. The ORF of human MCM7 was then amplified from cDNA of human fibroblasts (described in cell culture section). Capped RNA was transcribed from these plasmids after linearization using PmeI and using the T7 mMessage mMachine Kit (Ambion).

Casar Tena T., Maerz L.D., Szafranski K., Groth M., Blätte T.J., Donow C., Matysik S., Walther P., Jeggo P.A., Burkhalter M.D, & Philipp M. (2018). Resting cells rely on the DNA helicase component MCM2 to build cilia. Nucleic Acids Research, 47(1), 134-151.

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Plasmid Cloning and Transformation

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A list of constructs used in this study is provided in Supplementary Material. For cloning, PCR fragments were amplified using Platinum SuperFi II Polymerase (Invitrogen). The fragments were assembled by either NEBuilder HiFi Assembly (New England Biolabs, NEB) or TOPO directional cloning (Invitrogen). Assembled products were heat-shock transformed to 5-alpha (NEB), TOP10 (Invitrogen), or BL21 Star™ (DE3) (Invitrogen) bacteria.

Grady C.J., Castellanos Franco E.A., Schossau J., Ashbaugh R.C., Pelled G, & Gilad A.A. (2024). A putative design for the electromagnetic activation of split proteins for molecular and cellular manipulation. Frontiers in Bioengineering and Biotechnology, 12, 1355915.

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Plasmids for HDAC6 Functional Analysis

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Full-length FLAG epitope-tagged HDAC6 was a kind gift from Eric Verdin (Addgene plasmid #13823). The catalytically inactive mutant of HDAC6 (H216/611A, Addgene plasmid #30483) and C-terminal zinc finger domain (binder of ubiquitin zinc finger, BUZ Addgene plasmid #30484) deleted mutant were kind gifts from Tso-Pang Yao (Kawaguchi et al., 2003 (link)). All other constructs were cloned in-house by using topo-directional cloning (Invitrogen).

Nomura Y., Nakano M., Woo Sung H., Han M, & Pandey D. (2021). Inhibition of HDAC6 Activity Protects Against Endothelial Dysfunction and Atherogenesis in vivo: A Role for HDAC6 Neddylation. Frontiers in Physiology, 12, 675724.

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