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  • 97
    New England Biolabs bsmbi
    Synthetic assembly of the final multiplex hextuple luciferase vector (Basic protocol 3). ( A ) Overview of the in silico scarless assembly of a multiplex luciferase reporter in the destination vector Omega Destination-CMV:ELuc:bGH. Five “transcriptional reporter” plasmids, AlphaA 5xNF-KB:RedF:bGHpA (Addgene #124530) reporting on NF-κβ pathway signaling using red firefly luciferase (RF), AlphaB 7xSMAD:FLuc:bGHpA (Addgene #124531) reporting on TGF-β signaling using firefly luciferase (FL), AlphaC 3xDBE:Renilla:bGHpA (Addgene #124535) reporting on FoxO pathway signaling using renilla luciferase (Re), AlphaD 2xp53:NLuc:bGHpA (Addgene #124533) reporting on p53 pathway signaling using nano luciferase (NL), and AlphaE 6xAP1_RE:GrRenilla:bGHpA (Addgene #124534) reporting on MAPK/JNK pathway signaling using green renilla luciferase (GR), as well as the destination vector Omega Destination-CMV:ELuc:bGH containing the control enhanced beetle luciferase (EL) for normalization purposes are incubated together with <t>BsmBI</t> and T4 DNA ligase. Correct multipartitie stitching of all five BsmBI-released transcriptional luciferase reporter units into BsmBI-opended Omega Destination-CMV:ELuc:bGH, results in the final multiplex luciferase vector MLRV2:NF-kb-SMAD-DBE-P53-AP1 (Addgene #124536), consisting of five transcriptional luciferase reporter units stitched together in a specified order, and one control luciferase reporter unit (constitutively expressed ELuc luciferase). Assembled plasmids are identified as white colored colonies that are characterized further (see C ), while religated Omega Destination-CMV:ELuc:bGH plasmids are pink to purple colored due to the presence of the colorimetric marker, tinsel purple. ( B ) Overview of the cloning reaction. Prepare a reaction mix containing 75 ng of the final pink/white destination vector (Omega Destination-CMV:ELuc:bGH), 75 ng of each of the luciferase entry vectors, the type IIs restriction enzyme BsmBI, T4 DNA ligase and 10x T4 DNA ligase buffer. Start the assembly protocol that cycles 25 times between 37°C and 16°C. ( C ) Extended assembly cycling reaction conditions, with 50 cycles of 37°C and 16°C, followed by 1h at 37°C to favor digestion of uncut plasmids, 20 minutes at 85°C to denature the enzymes and a prolonged incubation at 16°C. ( D ) Restriction enzyme digestion of 3 white colored colonies using <t>ScaI</t> (6510, 2219, 1790, 1543, 1088 and 486 bps) and XhoI (6500, 2263, 1508, 1098, 979, 794 and 494 bps), and uncut DNA. All colonies show the correct digestion pattern.
    Bsmbi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsmbi/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bsmbi - by Bioz Stars, 2022-08
    97/100 stars
      Buy from Supplier

    96
    Thermo Fisher bsmbi
    Synthetic assembly of the final multiplex hextuple luciferase vector (Basic protocol 3). ( A ) Overview of the in silico scarless assembly of a multiplex luciferase reporter in the destination vector Omega Destination-CMV:ELuc:bGH. Five “transcriptional reporter” plasmids, AlphaA 5xNF-KB:RedF:bGHpA (Addgene #124530) reporting on NF-κβ pathway signaling using red firefly luciferase (RF), AlphaB 7xSMAD:FLuc:bGHpA (Addgene #124531) reporting on TGF-β signaling using firefly luciferase (FL), AlphaC 3xDBE:Renilla:bGHpA (Addgene #124535) reporting on FoxO pathway signaling using renilla luciferase (Re), AlphaD 2xp53:NLuc:bGHpA (Addgene #124533) reporting on p53 pathway signaling using nano luciferase (NL), and AlphaE 6xAP1_RE:GrRenilla:bGHpA (Addgene #124534) reporting on MAPK/JNK pathway signaling using green renilla luciferase (GR), as well as the destination vector Omega Destination-CMV:ELuc:bGH containing the control enhanced beetle luciferase (EL) for normalization purposes are incubated together with <t>BsmBI</t> and T4 DNA ligase. Correct multipartitie stitching of all five BsmBI-released transcriptional luciferase reporter units into BsmBI-opended Omega Destination-CMV:ELuc:bGH, results in the final multiplex luciferase vector MLRV2:NF-kb-SMAD-DBE-P53-AP1 (Addgene #124536), consisting of five transcriptional luciferase reporter units stitched together in a specified order, and one control luciferase reporter unit (constitutively expressed ELuc luciferase). Assembled plasmids are identified as white colored colonies that are characterized further (see C ), while religated Omega Destination-CMV:ELuc:bGH plasmids are pink to purple colored due to the presence of the colorimetric marker, tinsel purple. ( B ) Overview of the cloning reaction. Prepare a reaction mix containing 75 ng of the final pink/white destination vector (Omega Destination-CMV:ELuc:bGH), 75 ng of each of the luciferase entry vectors, the type IIs restriction enzyme BsmBI, T4 DNA ligase and 10x T4 DNA ligase buffer. Start the assembly protocol that cycles 25 times between 37°C and 16°C. ( C ) Extended assembly cycling reaction conditions, with 50 cycles of 37°C and 16°C, followed by 1h at 37°C to favor digestion of uncut plasmids, 20 minutes at 85°C to denature the enzymes and a prolonged incubation at 16°C. ( D ) Restriction enzyme digestion of 3 white colored colonies using <t>ScaI</t> (6510, 2219, 1790, 1543, 1088 and 486 bps) and XhoI (6500, 2263, 1508, 1098, 979, 794 and 494 bps), and uncut DNA. All colonies show the correct digestion pattern.
    Bsmbi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsmbi/product/Thermo Fisher
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bsmbi - by Bioz Stars, 2022-08
    96/100 stars
      Buy from Supplier

    95
    New England Biolabs bsmbi v2
    Synthetic assembly of the final multiplex hextuple luciferase vector (Basic protocol 3). ( A ) Overview of the in silico scarless assembly of a multiplex luciferase reporter in the destination vector Omega Destination-CMV:ELuc:bGH. Five “transcriptional reporter” plasmids, AlphaA 5xNF-KB:RedF:bGHpA (Addgene #124530) reporting on NF-κβ pathway signaling using red firefly luciferase (RF), AlphaB 7xSMAD:FLuc:bGHpA (Addgene #124531) reporting on TGF-β signaling using firefly luciferase (FL), AlphaC 3xDBE:Renilla:bGHpA (Addgene #124535) reporting on FoxO pathway signaling using renilla luciferase (Re), AlphaD 2xp53:NLuc:bGHpA (Addgene #124533) reporting on p53 pathway signaling using nano luciferase (NL), and AlphaE 6xAP1_RE:GrRenilla:bGHpA (Addgene #124534) reporting on MAPK/JNK pathway signaling using green renilla luciferase (GR), as well as the destination vector Omega Destination-CMV:ELuc:bGH containing the control enhanced beetle luciferase (EL) for normalization purposes are incubated together with <t>BsmBI</t> and T4 DNA ligase. Correct multipartitie stitching of all five BsmBI-released transcriptional luciferase reporter units into BsmBI-opended Omega Destination-CMV:ELuc:bGH, results in the final multiplex luciferase vector MLRV2:NF-kb-SMAD-DBE-P53-AP1 (Addgene #124536), consisting of five transcriptional luciferase reporter units stitched together in a specified order, and one control luciferase reporter unit (constitutively expressed ELuc luciferase). Assembled plasmids are identified as white colored colonies that are characterized further (see C ), while religated Omega Destination-CMV:ELuc:bGH plasmids are pink to purple colored due to the presence of the colorimetric marker, tinsel purple. ( B ) Overview of the cloning reaction. Prepare a reaction mix containing 75 ng of the final pink/white destination vector (Omega Destination-CMV:ELuc:bGH), 75 ng of each of the luciferase entry vectors, the type IIs restriction enzyme BsmBI, T4 DNA ligase and 10x T4 DNA ligase buffer. Start the assembly protocol that cycles 25 times between 37°C and 16°C. ( C ) Extended assembly cycling reaction conditions, with 50 cycles of 37°C and 16°C, followed by 1h at 37°C to favor digestion of uncut plasmids, 20 minutes at 85°C to denature the enzymes and a prolonged incubation at 16°C. ( D ) Restriction enzyme digestion of 3 white colored colonies using <t>ScaI</t> (6510, 2219, 1790, 1543, 1088 and 486 bps) and XhoI (6500, 2263, 1508, 1098, 979, 794 and 494 bps), and uncut DNA. All colonies show the correct digestion pattern.
    Bsmbi V2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsmbi v2/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bsmbi v2 - by Bioz Stars, 2022-08
    95/100 stars
      Buy from Supplier

    Image Search Results


    Synthetic assembly of the final multiplex hextuple luciferase vector (Basic protocol 3). ( A ) Overview of the in silico scarless assembly of a multiplex luciferase reporter in the destination vector Omega Destination-CMV:ELuc:bGH. Five “transcriptional reporter” plasmids, AlphaA 5xNF-KB:RedF:bGHpA (Addgene #124530) reporting on NF-κβ pathway signaling using red firefly luciferase (RF), AlphaB 7xSMAD:FLuc:bGHpA (Addgene #124531) reporting on TGF-β signaling using firefly luciferase (FL), AlphaC 3xDBE:Renilla:bGHpA (Addgene #124535) reporting on FoxO pathway signaling using renilla luciferase (Re), AlphaD 2xp53:NLuc:bGHpA (Addgene #124533) reporting on p53 pathway signaling using nano luciferase (NL), and AlphaE 6xAP1_RE:GrRenilla:bGHpA (Addgene #124534) reporting on MAPK/JNK pathway signaling using green renilla luciferase (GR), as well as the destination vector Omega Destination-CMV:ELuc:bGH containing the control enhanced beetle luciferase (EL) for normalization purposes are incubated together with BsmBI and T4 DNA ligase. Correct multipartitie stitching of all five BsmBI-released transcriptional luciferase reporter units into BsmBI-opended Omega Destination-CMV:ELuc:bGH, results in the final multiplex luciferase vector MLRV2:NF-kb-SMAD-DBE-P53-AP1 (Addgene #124536), consisting of five transcriptional luciferase reporter units stitched together in a specified order, and one control luciferase reporter unit (constitutively expressed ELuc luciferase). Assembled plasmids are identified as white colored colonies that are characterized further (see C ), while religated Omega Destination-CMV:ELuc:bGH plasmids are pink to purple colored due to the presence of the colorimetric marker, tinsel purple. ( B ) Overview of the cloning reaction. Prepare a reaction mix containing 75 ng of the final pink/white destination vector (Omega Destination-CMV:ELuc:bGH), 75 ng of each of the luciferase entry vectors, the type IIs restriction enzyme BsmBI, T4 DNA ligase and 10x T4 DNA ligase buffer. Start the assembly protocol that cycles 25 times between 37°C and 16°C. ( C ) Extended assembly cycling reaction conditions, with 50 cycles of 37°C and 16°C, followed by 1h at 37°C to favor digestion of uncut plasmids, 20 minutes at 85°C to denature the enzymes and a prolonged incubation at 16°C. ( D ) Restriction enzyme digestion of 3 white colored colonies using ScaI (6510, 2219, 1790, 1543, 1088 and 486 bps) and XhoI (6500, 2263, 1508, 1098, 979, 794 and 494 bps), and uncut DNA. All colonies show the correct digestion pattern.

    Journal: Current protocols in molecular biology

    Article Title: Rapid and efficient synthetic assembly of multiplex luciferase reporter plasmids for the simultaneous monitoring of up to six cellular signaling pathways

    doi: 10.1002/cpmb.121

    Figure Lengend Snippet: Synthetic assembly of the final multiplex hextuple luciferase vector (Basic protocol 3). ( A ) Overview of the in silico scarless assembly of a multiplex luciferase reporter in the destination vector Omega Destination-CMV:ELuc:bGH. Five “transcriptional reporter” plasmids, AlphaA 5xNF-KB:RedF:bGHpA (Addgene #124530) reporting on NF-κβ pathway signaling using red firefly luciferase (RF), AlphaB 7xSMAD:FLuc:bGHpA (Addgene #124531) reporting on TGF-β signaling using firefly luciferase (FL), AlphaC 3xDBE:Renilla:bGHpA (Addgene #124535) reporting on FoxO pathway signaling using renilla luciferase (Re), AlphaD 2xp53:NLuc:bGHpA (Addgene #124533) reporting on p53 pathway signaling using nano luciferase (NL), and AlphaE 6xAP1_RE:GrRenilla:bGHpA (Addgene #124534) reporting on MAPK/JNK pathway signaling using green renilla luciferase (GR), as well as the destination vector Omega Destination-CMV:ELuc:bGH containing the control enhanced beetle luciferase (EL) for normalization purposes are incubated together with BsmBI and T4 DNA ligase. Correct multipartitie stitching of all five BsmBI-released transcriptional luciferase reporter units into BsmBI-opended Omega Destination-CMV:ELuc:bGH, results in the final multiplex luciferase vector MLRV2:NF-kb-SMAD-DBE-P53-AP1 (Addgene #124536), consisting of five transcriptional luciferase reporter units stitched together in a specified order, and one control luciferase reporter unit (constitutively expressed ELuc luciferase). Assembled plasmids are identified as white colored colonies that are characterized further (see C ), while religated Omega Destination-CMV:ELuc:bGH plasmids are pink to purple colored due to the presence of the colorimetric marker, tinsel purple. ( B ) Overview of the cloning reaction. Prepare a reaction mix containing 75 ng of the final pink/white destination vector (Omega Destination-CMV:ELuc:bGH), 75 ng of each of the luciferase entry vectors, the type IIs restriction enzyme BsmBI, T4 DNA ligase and 10x T4 DNA ligase buffer. Start the assembly protocol that cycles 25 times between 37°C and 16°C. ( C ) Extended assembly cycling reaction conditions, with 50 cycles of 37°C and 16°C, followed by 1h at 37°C to favor digestion of uncut plasmids, 20 minutes at 85°C to denature the enzymes and a prolonged incubation at 16°C. ( D ) Restriction enzyme digestion of 3 white colored colonies using ScaI (6510, 2219, 1790, 1543, 1088 and 486 bps) and XhoI (6500, 2263, 1508, 1098, 979, 794 and 494 bps), and uncut DNA. All colonies show the correct digestion pattern.

    Article Snippet: Restriction enzymes: BsmBI (New England Biolabs, cat. no. R0580), ScaI-HF (New England Biolabs, cat. no. R3122), XhoI (New England Biolabs, cat. no. R0146).

    Techniques: Multiplex Assay, Luciferase, Plasmid Preparation, In Silico, Incubation, Marker, Clone Assay