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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+reactions/lr+clonase+reaction/pmc12264712-79-1-18
    Average 90 stars, based on 1 article reviews
    lr clonase reaction - by Bioz Stars, 2026-10
    90/100 stars

    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

    Drosophila:

    Article Title: Polycomb Requires Chaperonin Containing TCP-1 Subunit 7 for Maintaining Gene Silencing in Drosophila
    Article Snippet: Primers designed for Gateway Cloning were used to amplify CCT7 CDS from cDNA that was then cloned into pENTR/D-TOPO vector (Thermo Fisher Scientific, K240020). .. LR Clonase reactions (Thermo Fisher Scientific, 11791020) were set up with Drosophila Genomics Resource Center (DGRC) vectors for both cell culture and fly transformation containing either Myc or FLAG tags to prepare epitope-tagged CCT7. ..

    Cell Culture:

    Article Title: Polycomb Requires Chaperonin Containing TCP-1 Subunit 7 for Maintaining Gene Silencing in Drosophila
    Article Snippet: Primers designed for Gateway Cloning were used to amplify CCT7 CDS from cDNA that was then cloned into pENTR/D-TOPO vector (Thermo Fisher Scientific, K240020). .. LR Clonase reactions (Thermo Fisher Scientific, 11791020) were set up with Drosophila Genomics Resource Center (DGRC) vectors for both cell culture and fly transformation containing either Myc or FLAG tags to prepare epitope-tagged CCT7. ..

    Transformation Assay:

    Article Title: Polycomb Requires Chaperonin Containing TCP-1 Subunit 7 for Maintaining Gene Silencing in Drosophila
    Article Snippet: Primers designed for Gateway Cloning were used to amplify CCT7 CDS from cDNA that was then cloned into pENTR/D-TOPO vector (Thermo Fisher Scientific, K240020). .. LR Clonase reactions (Thermo Fisher Scientific, 11791020) were set up with Drosophila Genomics Resource Center (DGRC) vectors for both cell culture and fly transformation containing either Myc or FLAG tags to prepare epitope-tagged CCT7. ..

    Polymerase Chain Reaction:

    Article Title: Protein-folding chaperones predict structure-function relationships and cancer risk in BRCA1 mutation carriers
    Article Snippet: PCR products were purified using agarose gel extraction (Takara Bio, 740609), and BP clonase (Invitrogen, 11789) was used to insert BRCT fragments into the entry vector pDONR221. .. Next, two-step PCR-mediated site-directed mutagenesis was performed using Phusion High-Fidelity DNA Polymerase (New England BioLabs, M0530), and primers containing the mutation of interest were used to generate mutant BRCA1 fragments flanked by attL sequences for LR clonase reactions (Invitrogen, 11791). ..

    Article Title: Overexpression of ABI5 Binding Proteins Suppresses Inhibition of Germination Due to Overaccumulation of DELLA Proteins
    Article Snippet: .. Split YFP fusions for RGA, RGA2, and RGL2 were constructed using the Gateway compatible pSITE-cEYFP-C1 (Acc# GU734652) vector and PCR products with attL ends added as described in [ ], using primers described in , following manufacturer’s instructions for LR Clonase reactions (Invitrogen). ..

    Mutagenesis:

    Article Title: Protein-folding chaperones predict structure-function relationships and cancer risk in BRCA1 mutation carriers
    Article Snippet: PCR products were purified using agarose gel extraction (Takara Bio, 740609), and BP clonase (Invitrogen, 11789) was used to insert BRCT fragments into the entry vector pDONR221. .. Next, two-step PCR-mediated site-directed mutagenesis was performed using Phusion High-Fidelity DNA Polymerase (New England BioLabs, M0530), and primers containing the mutation of interest were used to generate mutant BRCA1 fragments flanked by attL sequences for LR clonase reactions (Invitrogen, 11791). ..

    Article Title: FAM122A ensures cell cycle interphase progression and checkpoint control as a SLiM-dependent substrate-competitive inhibitor to the B55⍺/PP2A phosphatase
    Article Snippet: .. Plasmids used or generated in this study are described in the Key resources table. pCMV6- Fam122a -Myc-DDK was purchased from Origene. pCMV6-AN-DDK- Fam122a was generated using SgfI/MluI restriction sites from the precision shuttle system (pCMV6-AN-DDK-Pol Iota-A kind gift from Roger Woodgate Addgene #131228). pCMV6-AN-mGFP and pCMV6-AN-mRFP plasmids were kind gifts from Richard Katz with Fam122a inserted using SgfI/MluI restriction sites. pGEX2T- Fam122a , pCMV6-AN-DDK- Fam122a , pCMV6-AN-mGFP- Fam122a , pCMV6-AN-RFP- Fam122a , pCW57.1-mRFP- Fam122a , and pGEX2T-FRs variant mutants were generated by site-directed mutagenesis using QuikChange II XL Site-directed mutagenesis (Agilent) with primers listed in the Appendix table and subsequently validated by Sanger sequencing. pGEX2T-FR deletion constructs were generated by PCR cloning BamHI/EcoRI restrictions sites, digestion, and ligation to pGEX2T. pCW57.1 FLAG-B55α and pCW57.1-mRFP- Fam122a were generated by LR clonase reactions (ThermoFisher Scientific) from the ORFs cloned in pENTR1A. (BamHI/EcoRI for mRFP- Fam122a and BglII/EcoRI for FLAG-B55α). ..

    Expressing:

    Article Title: Tuning SAS-6 architecture with monobodies impairs distinct steps of centriole assembly
    Article Snippet: .. Entry vectors were then used in LR Clonase reactions (ThermoFisher Scientific) with pCW57.1 (Addgene) to produce expression vectors. ..

    Cloning:

    Article Title: NTRK1 Fusion in Glioblastoma Multiforme
    Article Snippet: .. The NFASC-NTRK1 and EGFR vIII fragments were subcloned into an entry vector using pENTRTM/D-TOPO® Cloning Kit (Invitrogen), and then transferred into pLenti6.3/V5-DEST plasmid using LR clonase reactions (Invitrogen). .. The lentiviral vectors (pLKO.1-puro) harboring shRNAs targeting NTRK1 were purchased (Thermo).

    Plasmid Preparation:

    Article Title: NTRK1 Fusion in Glioblastoma Multiforme
    Article Snippet: .. The NFASC-NTRK1 and EGFR vIII fragments were subcloned into an entry vector using pENTRTM/D-TOPO® Cloning Kit (Invitrogen), and then transferred into pLenti6.3/V5-DEST plasmid using LR clonase reactions (Invitrogen). .. The lentiviral vectors (pLKO.1-puro) harboring shRNAs targeting NTRK1 were purchased (Thermo).

    Generated:

    Article Title: FAM122A ensures cell cycle interphase progression and checkpoint control as a SLiM-dependent substrate-competitive inhibitor to the B55⍺/PP2A phosphatase
    Article Snippet: .. Plasmids used or generated in this study are described in the Key resources table. pCMV6- Fam122a -Myc-DDK was purchased from Origene. pCMV6-AN-DDK- Fam122a was generated using SgfI/MluI restriction sites from the precision shuttle system (pCMV6-AN-DDK-Pol Iota-A kind gift from Roger Woodgate Addgene #131228). pCMV6-AN-mGFP and pCMV6-AN-mRFP plasmids were kind gifts from Richard Katz with Fam122a inserted using SgfI/MluI restriction sites. pGEX2T- Fam122a , pCMV6-AN-DDK- Fam122a , pCMV6-AN-mGFP- Fam122a , pCMV6-AN-RFP- Fam122a , pCW57.1-mRFP- Fam122a , and pGEX2T-FRs variant mutants were generated by site-directed mutagenesis using QuikChange II XL Site-directed mutagenesis (Agilent) with primers listed in the Appendix table and subsequently validated by Sanger sequencing. pGEX2T-FR deletion constructs were generated by PCR cloning BamHI/EcoRI restrictions sites, digestion, and ligation to pGEX2T. pCW57.1 FLAG-B55α and pCW57.1-mRFP- Fam122a were generated by LR clonase reactions (ThermoFisher Scientific) from the ORFs cloned in pENTR1A. (BamHI/EcoRI for mRFP- Fam122a and BglII/EcoRI for FLAG-B55α). ..

    Variant Assay:

    Article Title: FAM122A ensures cell cycle interphase progression and checkpoint control as a SLiM-dependent substrate-competitive inhibitor to the B55⍺/PP2A phosphatase
    Article Snippet: .. Plasmids used or generated in this study are described in the Key resources table. pCMV6- Fam122a -Myc-DDK was purchased from Origene. pCMV6-AN-DDK- Fam122a was generated using SgfI/MluI restriction sites from the precision shuttle system (pCMV6-AN-DDK-Pol Iota-A kind gift from Roger Woodgate Addgene #131228). pCMV6-AN-mGFP and pCMV6-AN-mRFP plasmids were kind gifts from Richard Katz with Fam122a inserted using SgfI/MluI restriction sites. pGEX2T- Fam122a , pCMV6-AN-DDK- Fam122a , pCMV6-AN-mGFP- Fam122a , pCMV6-AN-RFP- Fam122a , pCW57.1-mRFP- Fam122a , and pGEX2T-FRs variant mutants were generated by site-directed mutagenesis using QuikChange II XL Site-directed mutagenesis (Agilent) with primers listed in the Appendix table and subsequently validated by Sanger sequencing. pGEX2T-FR deletion constructs were generated by PCR cloning BamHI/EcoRI restrictions sites, digestion, and ligation to pGEX2T. pCW57.1 FLAG-B55α and pCW57.1-mRFP- Fam122a were generated by LR clonase reactions (ThermoFisher Scientific) from the ORFs cloned in pENTR1A. (BamHI/EcoRI for mRFP- Fam122a and BglII/EcoRI for FLAG-B55α). ..

    Sequencing:

    Article Title: FAM122A ensures cell cycle interphase progression and checkpoint control as a SLiM-dependent substrate-competitive inhibitor to the B55⍺/PP2A phosphatase
    Article Snippet: .. Plasmids used or generated in this study are described in the Key resources table. pCMV6- Fam122a -Myc-DDK was purchased from Origene. pCMV6-AN-DDK- Fam122a was generated using SgfI/MluI restriction sites from the precision shuttle system (pCMV6-AN-DDK-Pol Iota-A kind gift from Roger Woodgate Addgene #131228). pCMV6-AN-mGFP and pCMV6-AN-mRFP plasmids were kind gifts from Richard Katz with Fam122a inserted using SgfI/MluI restriction sites. pGEX2T- Fam122a , pCMV6-AN-DDK- Fam122a , pCMV6-AN-mGFP- Fam122a , pCMV6-AN-RFP- Fam122a , pCW57.1-mRFP- Fam122a , and pGEX2T-FRs variant mutants were generated by site-directed mutagenesis using QuikChange II XL Site-directed mutagenesis (Agilent) with primers listed in the Appendix table and subsequently validated by Sanger sequencing. pGEX2T-FR deletion constructs were generated by PCR cloning BamHI/EcoRI restrictions sites, digestion, and ligation to pGEX2T. pCW57.1 FLAG-B55α and pCW57.1-mRFP- Fam122a were generated by LR clonase reactions (ThermoFisher Scientific) from the ORFs cloned in pENTR1A. (BamHI/EcoRI for mRFP- Fam122a and BglII/EcoRI for FLAG-B55α). ..

    Construct:

    Article Title: FAM122A ensures cell cycle interphase progression and checkpoint control as a SLiM-dependent substrate-competitive inhibitor to the B55⍺/PP2A phosphatase
    Article Snippet: .. Plasmids used or generated in this study are described in the Key resources table. pCMV6- Fam122a -Myc-DDK was purchased from Origene. pCMV6-AN-DDK- Fam122a was generated using SgfI/MluI restriction sites from the precision shuttle system (pCMV6-AN-DDK-Pol Iota-A kind gift from Roger Woodgate Addgene #131228). pCMV6-AN-mGFP and pCMV6-AN-mRFP plasmids were kind gifts from Richard Katz with Fam122a inserted using SgfI/MluI restriction sites. pGEX2T- Fam122a , pCMV6-AN-DDK- Fam122a , pCMV6-AN-mGFP- Fam122a , pCMV6-AN-RFP- Fam122a , pCW57.1-mRFP- Fam122a , and pGEX2T-FRs variant mutants were generated by site-directed mutagenesis using QuikChange II XL Site-directed mutagenesis (Agilent) with primers listed in the Appendix table and subsequently validated by Sanger sequencing. pGEX2T-FR deletion constructs were generated by PCR cloning BamHI/EcoRI restrictions sites, digestion, and ligation to pGEX2T. pCW57.1 FLAG-B55α and pCW57.1-mRFP- Fam122a were generated by LR clonase reactions (ThermoFisher Scientific) from the ORFs cloned in pENTR1A. (BamHI/EcoRI for mRFP- Fam122a and BglII/EcoRI for FLAG-B55α). ..

    Article Title: Overexpression of ABI5 Binding Proteins Suppresses Inhibition of Germination Due to Overaccumulation of DELLA Proteins
    Article Snippet: .. Split YFP fusions for RGA, RGA2, and RGL2 were constructed using the Gateway compatible pSITE-cEYFP-C1 (Acc# GU734652) vector and PCR products with attL ends added as described in [ ], using primers described in , following manufacturer’s instructions for LR Clonase reactions (Invitrogen). ..

    other:

    Article Title: Completing the TRB family: newly characterized members show ancient evolutionary origins and distinct localization, yet similar interactions
    Article Snippet: For protein expression in Escherichia coli , constructs in pDONR207 (for TRB1 and TRB5) or in pENTR223 (for TRB4) were used as donor vectors for LR ClonaseTM reactions (Thermo Fisher Scientific), where genes coding proteins of interest (TRB1, 4 and 5) were transferred into the destination vector pHGWA ( ).



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    (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