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lr clonase reaction  (Thermo Fisher)


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    Structured Review

    Thermo Fisher lr clonase reaction
    (A) LR <t>clonase</t> reaction used to insert the Six4 enhancer sequence upstream of the nuclear red fluorescent protein sequence, mCherry:Renilla(nls), in the Vanglow#83339 plasmid. Entry clone and toxic byproduct plasmids are Kanamycin resistant (KanR), and destination vector and expression clone are Ampicillin resistant (AmpR). The construct injected is labeled as expression clone. (B) Sequence map of the plasmid generated to develop the six4 nlsRFP reporter. The Six4 enhancer noted on the map is the third intron of the D- Six4 gene sequence, diagrammed below the plasmid (exons, orange; UTRs, gray; introns, horizontal line). (C-H) Whole embryo, fixed tissue immunostaining for RFP to show six4 nlsRFP reporter expression for the construct injected on chromosomes III (C-E) and II (F-H) at embryonic stages 11 (C,F) , 15-16 (D,G) , and 17 (E,H) with the gonad outlined by yellow boxes. Expression of Six4 in the ventral nervous system (H, yellow arrow). Prime panels show high magnification of gonads in corresponding panels, outlined with dotted white line. RFP, red; Fas3, white. ( I-M ) Live imaging of stem cell niche assembly during embryonic stages 15-17 using the six4 nlsRFP reporter (magenta) to show SGP nuclei. Two individual niche cells over time (I-I'''', yellow arrows). Gonad outlined in white. Niche within gonad in I'''' also outlined in white. Time indicated in hh:mm:ss.
    Lr Clonase Reaction, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/lr+clonase+reaction/lr+clonase+reaction/pmc12264712-79-1-18
    Average 90 stars, based on 1 article reviews
    lr clonase reaction - by Bioz Stars, 2026-09
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    Images

    1) Product Images from "Generation of six4 -nlsRFP: a red somatic gonadal nuclear marker for live imaging Drosophila gonadogenesis"

    Article Title: Generation of six4 -nlsRFP: a red somatic gonadal nuclear marker for live imaging Drosophila gonadogenesis

    Journal: microPublication Biology

    doi: 10.17912/micropub.biology.001652

    (A) LR clonase reaction used to insert the Six4 enhancer sequence upstream of the nuclear red fluorescent protein sequence, mCherry:Renilla(nls), in the Vanglow#83339 plasmid. Entry clone and toxic byproduct plasmids are Kanamycin resistant (KanR), and destination vector and expression clone are Ampicillin resistant (AmpR). The construct injected is labeled as expression clone. (B) Sequence map of the plasmid generated to develop the six4 nlsRFP reporter. The Six4 enhancer noted on the map is the third intron of the D- Six4 gene sequence, diagrammed below the plasmid (exons, orange; UTRs, gray; introns, horizontal line). (C-H) Whole embryo, fixed tissue immunostaining for RFP to show six4 nlsRFP reporter expression for the construct injected on chromosomes III (C-E) and II (F-H) at embryonic stages 11 (C,F) , 15-16 (D,G) , and 17 (E,H) with the gonad outlined by yellow boxes. Expression of Six4 in the ventral nervous system (H, yellow arrow). Prime panels show high magnification of gonads in corresponding panels, outlined with dotted white line. RFP, red; Fas3, white. ( I-M ) Live imaging of stem cell niche assembly during embryonic stages 15-17 using the six4 nlsRFP reporter (magenta) to show SGP nuclei. Two individual niche cells over time (I-I'''', yellow arrows). Gonad outlined in white. Niche within gonad in I'''' also outlined in white. Time indicated in hh:mm:ss.
    Figure Legend Snippet: (A) LR clonase reaction used to insert the Six4 enhancer sequence upstream of the nuclear red fluorescent protein sequence, mCherry:Renilla(nls), in the Vanglow#83339 plasmid. Entry clone and toxic byproduct plasmids are Kanamycin resistant (KanR), and destination vector and expression clone are Ampicillin resistant (AmpR). The construct injected is labeled as expression clone. (B) Sequence map of the plasmid generated to develop the six4 nlsRFP reporter. The Six4 enhancer noted on the map is the third intron of the D- Six4 gene sequence, diagrammed below the plasmid (exons, orange; UTRs, gray; introns, horizontal line). (C-H) Whole embryo, fixed tissue immunostaining for RFP to show six4 nlsRFP reporter expression for the construct injected on chromosomes III (C-E) and II (F-H) at embryonic stages 11 (C,F) , 15-16 (D,G) , and 17 (E,H) with the gonad outlined by yellow boxes. Expression of Six4 in the ventral nervous system (H, yellow arrow). Prime panels show high magnification of gonads in corresponding panels, outlined with dotted white line. RFP, red; Fas3, white. ( I-M ) Live imaging of stem cell niche assembly during embryonic stages 15-17 using the six4 nlsRFP reporter (magenta) to show SGP nuclei. Two individual niche cells over time (I-I'''', yellow arrows). Gonad outlined in white. Niche within gonad in I'''' also outlined in white. Time indicated in hh:mm:ss.

    Techniques Used: Sequencing, Plasmid Preparation, Expressing, Construct, Injection, Labeling, Generated, Immunostaining, Imaging

    Related Articles

    Plasmid Preparation:

    Article Title: Identification of parthenogenesis-inducing effector proteins in Wolbachia
    Article Snippet: Entry vectors were verified first with restriction digests, followed by whole-plasmid sequencing (Plasmidsaurus). .. Validated entry vectors were recombined into an appropriate destination vector (see Supplemental Table S3) in an LR clonase reaction following manufacturer’s instructions (Invitrogen). .. Expression vectors were cloned into One Shot Top10 competent cells (Invitrogen), plated on selective media, and validated as above.

    Article Title: The transcription factor PpRKD evokes female developmental fate in the sexual reproductive organs of Physcomitrium patens.
    Article Snippet: To generate a Citrine-fusion type entry vector (pENTR_PpRKDir_Citrine), the DNA fragments produced by a NotI–BssHII double digestion of pENTR_PpRKDir and pENTR-Citrine were ligated together using T4 DNA ligase (Toyobo). .. The respective entry constructs were recombined into the Gateway site of the pPGX8 plasmid (AB537482) (Kubo et al., 2013) or pPGX8DR (Do et al., 2020) via an LR Clonase reaction (ThermoFisher Scientific). ..

    Article Title: Phloem-Mobile MYB44 Negatively Regulates Expression of PHOSPHATE TRANSPORTER 1 in Arabidopsis Roots.
    Article Snippet: .. The fragment was then inserted into the TOPO-D vector (Thermo Fisher Scientific, Waltham, MA, USA), followed by an LR clonase reaction (Invitrogen), to subclone the AtMYB44 promoter into pBGWFS7. ..

    Expressing:

    Article Title: Novel Nuclear Roles for Testis-Specific ACTL7A and ACTL7B Supported by In Vivo Characterizations and AI Facilitated In Silico Mechanistic Modeling with Implications for Epigenetic Regulation in Spermiogenesis
    Article Snippet: The process entailed cloning the various human ACTL7B gene variants into the pENTR TOPO vectors using the pENTR d-Topo cloning kit (Invitrogen), following the manufacturer’s guidelines. .. The full-length ACTL7B expression vector as well as its truncated variants were generated through LR clonase reaction (Gateway® LR clonaseTM II Enzyme Mix, Invitrogen, Waltham, MA, USA) using the destination vector pcDNA6.2/N-YFP-DEST (Supplementary Fig. S1A-F). ..

    Generated:

    Article Title: Novel Nuclear Roles for Testis-Specific ACTL7A and ACTL7B Supported by In Vivo Characterizations and AI Facilitated In Silico Mechanistic Modeling with Implications for Epigenetic Regulation in Spermiogenesis
    Article Snippet: The process entailed cloning the various human ACTL7B gene variants into the pENTR TOPO vectors using the pENTR d-Topo cloning kit (Invitrogen), following the manufacturer’s guidelines. .. The full-length ACTL7B expression vector as well as its truncated variants were generated through LR clonase reaction (Gateway® LR clonaseTM II Enzyme Mix, Invitrogen, Waltham, MA, USA) using the destination vector pcDNA6.2/N-YFP-DEST (Supplementary Fig. S1A-F). ..

    Construct:

    Article Title: The transcription factor PpRKD evokes female developmental fate in the sexual reproductive organs of Physcomitrium patens.
    Article Snippet: To generate a Citrine-fusion type entry vector (pENTR_PpRKDir_Citrine), the DNA fragments produced by a NotI–BssHII double digestion of pENTR_PpRKDir and pENTR-Citrine were ligated together using T4 DNA ligase (Toyobo). .. The respective entry constructs were recombined into the Gateway site of the pPGX8 plasmid (AB537482) (Kubo et al., 2013) or pPGX8DR (Do et al., 2020) via an LR Clonase reaction (ThermoFisher Scientific). ..

    Clone Assay:

    Article Title: Phosphorylation-linked complex profiling identifies assemblies required for Hippo signal integration
    Article Snippet: .. The integration of the entry clones into the Gateway destination vectors (pcDNA5/ FRT/TO/SH/GW; Glatter et al, 2009) was performed with an enzymatic LR clonase reaction (Invitrogen). .. YAP1 mutant sequences were generated by gene synthesis in pDONR223 (Biocat GMBH) and subsequently cloned into pcDNA5/FRT/TO/SH/GW as described above.

    Article Title: StCDF1 : A ‘ jack of all trades ’ clock output with a central role in regulating potato nitrate reduction activity
    Article Snippet: .. The PCR amplification products were cloned into the GATEWAY cloning vector pDONR207 and later inserted via the LR clonase reaction (Invitrogen) in front of the luciferase reporter in the pGWB435 destination vector. .. To avoid background activity originated by basal promoter expression in Agrobacteria, a modified version of this vector (pGWBiLUC), bearing the potato L700 intron inserted into the LUC coding region was used in these studies.

    Polymerase Chain Reaction:

    Article Title: StCDF1 : A ‘ jack of all trades ’ clock output with a central role in regulating potato nitrate reduction activity
    Article Snippet: .. The PCR amplification products were cloned into the GATEWAY cloning vector pDONR207 and later inserted via the LR clonase reaction (Invitrogen) in front of the luciferase reporter in the pGWB435 destination vector. .. To avoid background activity originated by basal promoter expression in Agrobacteria, a modified version of this vector (pGWBiLUC), bearing the potato L700 intron inserted into the LUC coding region was used in these studies.

    Article Title: Identification of host specificity determinants in brome mosaic virus for rice infection.
    Article Snippet: .. After PCR amplification using pB2KU52 or pB2KU52776R+784T as a template, the amplified fragments were transferred into pBTM116-GW by the Gateway system using an LR clonase reaction with the pENTR/D-TOPO cloning kit (Thermo Fisher Scientific). ..

    Amplification:

    Article Title: StCDF1 : A ‘ jack of all trades ’ clock output with a central role in regulating potato nitrate reduction activity
    Article Snippet: .. The PCR amplification products were cloned into the GATEWAY cloning vector pDONR207 and later inserted via the LR clonase reaction (Invitrogen) in front of the luciferase reporter in the pGWB435 destination vector. .. To avoid background activity originated by basal promoter expression in Agrobacteria, a modified version of this vector (pGWBiLUC), bearing the potato L700 intron inserted into the LUC coding region was used in these studies.

    Article Title: Identification of host specificity determinants in brome mosaic virus for rice infection.
    Article Snippet: .. After PCR amplification using pB2KU52 or pB2KU52776R+784T as a template, the amplified fragments were transferred into pBTM116-GW by the Gateway system using an LR clonase reaction with the pENTR/D-TOPO cloning kit (Thermo Fisher Scientific). ..

    Cloning:

    Article Title: StCDF1 : A ‘ jack of all trades ’ clock output with a central role in regulating potato nitrate reduction activity
    Article Snippet: .. The PCR amplification products were cloned into the GATEWAY cloning vector pDONR207 and later inserted via the LR clonase reaction (Invitrogen) in front of the luciferase reporter in the pGWB435 destination vector. .. To avoid background activity originated by basal promoter expression in Agrobacteria, a modified version of this vector (pGWBiLUC), bearing the potato L700 intron inserted into the LUC coding region was used in these studies.

    Article Title: Identification of host specificity determinants in brome mosaic virus for rice infection.
    Article Snippet: .. After PCR amplification using pB2KU52 or pB2KU52776R+784T as a template, the amplified fragments were transferred into pBTM116-GW by the Gateway system using an LR clonase reaction with the pENTR/D-TOPO cloning kit (Thermo Fisher Scientific). ..

    Luciferase:

    Article Title: StCDF1 : A ‘ jack of all trades ’ clock output with a central role in regulating potato nitrate reduction activity
    Article Snippet: .. The PCR amplification products were cloned into the GATEWAY cloning vector pDONR207 and later inserted via the LR clonase reaction (Invitrogen) in front of the luciferase reporter in the pGWB435 destination vector. .. To avoid background activity originated by basal promoter expression in Agrobacteria, a modified version of this vector (pGWBiLUC), bearing the potato L700 intron inserted into the LUC coding region was used in these studies.



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    Image Search Results


    (A) LR clonase reaction used to insert the Six4 enhancer sequence upstream of the nuclear red fluorescent protein sequence, mCherry:Renilla(nls), in the Vanglow#83339 plasmid. Entry clone and toxic byproduct plasmids are Kanamycin resistant (KanR), and destination vector and expression clone are Ampicillin resistant (AmpR). The construct injected is labeled as expression clone. (B) Sequence map of the plasmid generated to develop the six4 nlsRFP reporter. The Six4 enhancer noted on the map is the third intron of the D- Six4 gene sequence, diagrammed below the plasmid (exons, orange; UTRs, gray; introns, horizontal line). (C-H) Whole embryo, fixed tissue immunostaining for RFP to show six4 nlsRFP reporter expression for the construct injected on chromosomes III (C-E) and II (F-H) at embryonic stages 11 (C,F) , 15-16 (D,G) , and 17 (E,H) with the gonad outlined by yellow boxes. Expression of Six4 in the ventral nervous system (H, yellow arrow). Prime panels show high magnification of gonads in corresponding panels, outlined with dotted white line. RFP, red; Fas3, white. ( I-M ) Live imaging of stem cell niche assembly during embryonic stages 15-17 using the six4 nlsRFP reporter (magenta) to show SGP nuclei. Two individual niche cells over time (I-I'''', yellow arrows). Gonad outlined in white. Niche within gonad in I'''' also outlined in white. Time indicated in hh:mm:ss.

    Journal: microPublication Biology

    Article Title: Generation of six4 -nlsRFP: a red somatic gonadal nuclear marker for live imaging Drosophila gonadogenesis

    doi: 10.17912/micropub.biology.001652

    Figure Lengend Snippet: (A) LR clonase reaction used to insert the Six4 enhancer sequence upstream of the nuclear red fluorescent protein sequence, mCherry:Renilla(nls), in the Vanglow#83339 plasmid. Entry clone and toxic byproduct plasmids are Kanamycin resistant (KanR), and destination vector and expression clone are Ampicillin resistant (AmpR). The construct injected is labeled as expression clone. (B) Sequence map of the plasmid generated to develop the six4 nlsRFP reporter. The Six4 enhancer noted on the map is the third intron of the D- Six4 gene sequence, diagrammed below the plasmid (exons, orange; UTRs, gray; introns, horizontal line). (C-H) Whole embryo, fixed tissue immunostaining for RFP to show six4 nlsRFP reporter expression for the construct injected on chromosomes III (C-E) and II (F-H) at embryonic stages 11 (C,F) , 15-16 (D,G) , and 17 (E,H) with the gonad outlined by yellow boxes. Expression of Six4 in the ventral nervous system (H, yellow arrow). Prime panels show high magnification of gonads in corresponding panels, outlined with dotted white line. RFP, red; Fas3, white. ( I-M ) Live imaging of stem cell niche assembly during embryonic stages 15-17 using the six4 nlsRFP reporter (magenta) to show SGP nuclei. Two individual niche cells over time (I-I'''', yellow arrows). Gonad outlined in white. Niche within gonad in I'''' also outlined in white. Time indicated in hh:mm:ss.

    Article Snippet: An LR clonase reaction was performed to induce recombination at the attL and attR sites between these plasmids (ThermoFisher Scientific).

    Techniques: Sequencing, Plasmid Preparation, Expressing, Construct, Injection, Labeling, Generated, Immunostaining, Imaging