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Pentr 5 topo vector

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The PENTR-5'-TOPO vector is a cloning vector designed for the direct insertion of PCR products. It facilitates the efficient and rapid cloning of blunt-ended DNA fragments into an expression vector. The vector contains the TOPO recognition site, which enables the directional cloning of PCR products without the need for restriction enzyme digestion or ligation.

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Lab products found in correlation

Pentr d topo vector, by Thermo Fisher Scientific (2 mentions) Blasticidin, by InvivoGen (2 mentions)

Spelling variants of this product

TOPO vector (87 citations) PENTR vector (44 citations) PENTR-TOPO (16 citations) PENTR-TOPO vector (8 citations)

3 protocols using pentr 5 topo vector

1

Cloning Human Enhancer Variants

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pRL049 Hs_enh9Bwt was amplified with the following primers, using human genomic DNA as template, and then cloned into the pENTR5’-TOPO vector (Thermo Fisher #K59120):
oRL163 Hs_enh9B Fw: 5’-AGTAGGGGGATGCTAATTTCATAGC-3’
oRL164 Hs_enh9B Rev: 5’-TCTCAAGTCCTCTTGCGGTTTTGA-3’
pRL051 Hs_enh9T>G was cloned using pRL049 as a template for overlapping PCR to introduce the T>G mutation at base pair position 339 using the following primers:
oRL165 Hs_enh9B overlap Rev: 5’-CCCAACAGTTTCCAAGCATATTGAATATATTGTGGTCAG-3’
oRL166 Hs_enh9B overlap Fw: 5’-CTGACCACAATATATTCCATATTCTTGGAAACTGTTGGG-3’
The products of oRL163 Fw + oRL65 Rev and oRL166 Fw + oRL164 Rev were individually column-purified and 1 μl of each was combined as template for a final PCR reaction using oRL163 Fw + oRL164 Rev. The final product was then cloned into the pENTR5’-TOPO vector (Thermo Fisher #K59120).
Lalonde R.L., Wells H.H., Kemmler C.L., Nieuwenhuize S., Lerma R., Burger A, & Mosimann C. (2023). pIGLET: Safe harbor landing sites for reproducible and efficient transgenesis in zebrafish. bioRxiv.
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2

Lentiviral Construct Generation for Circadian Regulation

Cited 1 time
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Bmal1 and UBC promoters were cloned into the pENTR-5’-TOPO vector (Life Technologies) as before17 (link). Mouse Bmal1, Bmal2 and various chimeras or mutants were first subcloned to p3xFlag-CMV-10 or -14 vectors to obtain 3xFlag tags at the N- or C-terminus, respectively, and then cloned to the pENTR/D-TOPO vector (Life Technologies). The promoter and cDNA pENTR vectors were recombined with pLV7 destination vector56 using Clonase (Life Technologies) to generate the lentiviral expression constructs. All constructs were verified by sequencing of the entire open reading frame.
Recombinant lentiviral particles were produced by transient transfection in human embryonic kidney HEK293T cells (ATCC) using the calcium-phosphate method as previously described56 ,57 (link). Bmal1–/–Per2Luc mouse fibroblast cell line was generated in our previous studies17 (link). Cells were cultured in DMEM supplemented with 10% FBS and 1x penicillin-streptomycin-glutamine mixture. All cell culture reagents were from HyClone. For infection of Bmal1–/–Per2Luc fibroblasts, culture medium containing viral particles (~106 viral particles/ml) were harvested at 48 hr post-transfection and used for subsequent infection of cells. Transduced cells were selected with 10 μg/ml blasticidin (InvivoGen) as previously done17 (link).
Xu H., Gustafson C.L., Sammons P.J., Khan S.K., Parsley N.C., Ramanathan C., Lee H.W., Liu A.C, & Partch C.L. (2015). Cryptochrome 1 regulates the circadian clock through dynamic interactions with the BMAL1 C-terminus. Nature structural & molecular biology, 22(6), 476-484.
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3

Lentiviral Construct Generation for Circadian Regulation

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Bmal1 and UBC promoters were cloned into the pENTR-5’-TOPO vector (Life Technologies) as before17 (link). Mouse Bmal1, Bmal2 and various chimeras or mutants were first subcloned to p3xFlag-CMV-10 or -14 vectors to obtain 3xFlag tags at the N- or C-terminus, respectively, and then cloned to the pENTR/D-TOPO vector (Life Technologies). The promoter and cDNA pENTR vectors were recombined with pLV7 destination vector56 using Clonase (Life Technologies) to generate the lentiviral expression constructs. All constructs were verified by sequencing of the entire open reading frame.
Recombinant lentiviral particles were produced by transient transfection in human embryonic kidney HEK293T cells (ATCC) using the calcium-phosphate method as previously described56 ,57 (link). Bmal1–/–Per2Luc mouse fibroblast cell line was generated in our previous studies17 (link). Cells were cultured in DMEM supplemented with 10% FBS and 1x penicillin-streptomycin-glutamine mixture. All cell culture reagents were from HyClone. For infection of Bmal1–/–Per2Luc fibroblasts, culture medium containing viral particles (~106 viral particles/ml) were harvested at 48 hr post-transfection and used for subsequent infection of cells. Transduced cells were selected with 10 μg/ml blasticidin (InvivoGen) as previously done17 (link).
Xu H., Gustafson C.L., Sammons P.J., Khan S.K., Parsley N.C., Ramanathan C., Lee H.W., Liu A.C, & Partch C.L. (2015). Cryptochrome 1 regulates the circadian clock through dynamic interactions with the BMAL1 C-terminus. Nature structural & molecular biology, 22(6), 476-484.
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