bbsi  (New England Biolabs)


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    BbsI
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    BbsI 1 500 units
    Catalog Number:
    R0539L
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    Category:
    Restriction Enzymes
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    1 500 units
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    New England Biolabs bbsi
    BbsI
    BbsI 1 500 units
    https://www.bioz.com/result/bbsi/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bbsi - by Bioz Stars, 2021-06
    99/100 stars

    Images

    1) Product Images from "Preparation of Group I Introns for Biochemical Studies and Crystallization Assays by Native Affinity Purification"

    Article Title: Preparation of Group I Introns for Biochemical Studies and Crystallization Assays by Native Affinity Purification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006740

    A general strategy to synthesize and purify group I introns within the corresponding exons. (A) General scheme of synthesis by PCR from overlapping primers followed by native purification of the transcribed RNA. The variant shown contains a group I intron (red), 5′ and 3′ exons (purple), a T7 promoter (blue), and restriction sites suitable for cloning (green). The mutated HDV, the dual SRP tag, and the Tma M protein coupled to a support are shown in orange, black, and cyan, respectively. Note that the NcoI restriction site was engineered within the 5′ end of the HDV mutant [3] , while the BbsI restriction site is no longer present in the final RNA. (B) Affinity purification chart for small-scale transcription reactions developed in this study (WB: wash buffer; CB: cleavage buffer; SB: storage buffer; RB: regeneration buffer; see Materials and Methods section).
    Figure Legend Snippet: A general strategy to synthesize and purify group I introns within the corresponding exons. (A) General scheme of synthesis by PCR from overlapping primers followed by native purification of the transcribed RNA. The variant shown contains a group I intron (red), 5′ and 3′ exons (purple), a T7 promoter (blue), and restriction sites suitable for cloning (green). The mutated HDV, the dual SRP tag, and the Tma M protein coupled to a support are shown in orange, black, and cyan, respectively. Note that the NcoI restriction site was engineered within the 5′ end of the HDV mutant [3] , while the BbsI restriction site is no longer present in the final RNA. (B) Affinity purification chart for small-scale transcription reactions developed in this study (WB: wash buffer; CB: cleavage buffer; SB: storage buffer; RB: regeneration buffer; see Materials and Methods section).

    Techniques Used: Polymerase Chain Reaction, Purification, Variant Assay, Clone Assay, Mutagenesis, Affinity Purification, Western Blot

    2) Product Images from "Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis"

    Article Title: Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14329-5

    Architecture of the MCS-IIS C2. This DNA-sequence is located on the cargo vector between the E. coli ori and the Bacillus antibiotic marker. The recognition sites for five type IIS restriction enzymes (AarI, BtgZI, BbsI, BsaI, BsmBI), each designed to create a 5′ GCGA-overhang are encoded on the DNA stretch. Architecture of all MCS-IIS can be found in Fig. S1 .
    Figure Legend Snippet: Architecture of the MCS-IIS C2. This DNA-sequence is located on the cargo vector between the E. coli ori and the Bacillus antibiotic marker. The recognition sites for five type IIS restriction enzymes (AarI, BtgZI, BbsI, BsaI, BsmBI), each designed to create a 5′ GCGA-overhang are encoded on the DNA stretch. Architecture of all MCS-IIS can be found in Fig. S1 .

    Techniques Used: Sequencing, Plasmid Preparation, Marker

    3) Product Images from "Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis"

    Article Title: Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14329-5

    Architecture of the MCS-IIS C2. This DNA-sequence is located on the cargo vector between the E. coli ori and the Bacillus antibiotic marker. The recognition sites for five type IIS restriction enzymes (AarI, BtgZI, BbsI, BsaI, BsmBI), each designed to create a 5′ GCGA-overhang are encoded on the DNA stretch. Architecture of all MCS-IIS can be found in Fig. S1 .
    Figure Legend Snippet: Architecture of the MCS-IIS C2. This DNA-sequence is located on the cargo vector between the E. coli ori and the Bacillus antibiotic marker. The recognition sites for five type IIS restriction enzymes (AarI, BtgZI, BbsI, BsaI, BsmBI), each designed to create a 5′ GCGA-overhang are encoded on the DNA stretch. Architecture of all MCS-IIS can be found in Fig. S1 .

    Techniques Used: Sequencing, Plasmid Preparation, Marker

    4) Product Images from "Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory"

    Article Title: Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20051155

    Overview of the experimental design for CiPDS disruption. ( A ) Schematic position of the two guide RNAs (red boxes) targeting the fifth exon of CiPDS . The blue boxes indicate exons; the black lines indicate introns. ( B ) Schematic view of pKanCiU6-1p-sgRNAscaffold. Between the CiU6-1p and the sgRNA scaffold (scaff), a ccdB gene driven by the LacZ promoter (counter selection marker) is surrounded by BbsI (second-generation enzyme) recognition sites, which allows the ligation of the hybridized guide adaptor shown in C . Red B-L depicts the orientation of the BbsI recognition site which achieves the cleavage on the left side. Blue B-R depicts the orientation of the BbsI recognition site which achieves cleavage on the right side. ( C ) Sequences of the complementary guide adaptors with the 19 pb guide sequence (yellow), the transcription initiator nucleotide G (bold), and the binding sites necessary to insert the hybridized guide adaptor into the pKanCiU6-1p-sgRNAscaffold (red). ( D ) Representation of the vector resulting from the insertion of the hybridized guide adaptors in the pKanCiU6-1p-sgRNAscaffold, (note that one vector is constructed for each guide). The primers used for preparation of CiU6-1p-guide-sgRNA cassettes for Golden Gate Cloning are also shown. ( E ) Representation of the final plasmid pYLCRISPR-sgRNA1-sgRNA2 resulting from the cloning of the two cassettes into the pYLCRISPR/Cas9P 35S -B [ 36 ]. RB = Right Border, LB = Left Border, NLS = Nuclear Localization Signal.
    Figure Legend Snippet: Overview of the experimental design for CiPDS disruption. ( A ) Schematic position of the two guide RNAs (red boxes) targeting the fifth exon of CiPDS . The blue boxes indicate exons; the black lines indicate introns. ( B ) Schematic view of pKanCiU6-1p-sgRNAscaffold. Between the CiU6-1p and the sgRNA scaffold (scaff), a ccdB gene driven by the LacZ promoter (counter selection marker) is surrounded by BbsI (second-generation enzyme) recognition sites, which allows the ligation of the hybridized guide adaptor shown in C . Red B-L depicts the orientation of the BbsI recognition site which achieves the cleavage on the left side. Blue B-R depicts the orientation of the BbsI recognition site which achieves cleavage on the right side. ( C ) Sequences of the complementary guide adaptors with the 19 pb guide sequence (yellow), the transcription initiator nucleotide G (bold), and the binding sites necessary to insert the hybridized guide adaptor into the pKanCiU6-1p-sgRNAscaffold (red). ( D ) Representation of the vector resulting from the insertion of the hybridized guide adaptors in the pKanCiU6-1p-sgRNAscaffold, (note that one vector is constructed for each guide). The primers used for preparation of CiU6-1p-guide-sgRNA cassettes for Golden Gate Cloning are also shown. ( E ) Representation of the final plasmid pYLCRISPR-sgRNA1-sgRNA2 resulting from the cloning of the two cassettes into the pYLCRISPR/Cas9P 35S -B [ 36 ]. RB = Right Border, LB = Left Border, NLS = Nuclear Localization Signal.

    Techniques Used: Selection, Marker, Ligation, Sequencing, Binding Assay, Plasmid Preparation, Construct, Clone Assay

    5) Product Images from "90-Kilodalton Heat Shock Protein, Hsp90, as a Target for Genotyping Cryptosporidium spp. Known To Infect Humans ▿ spp. Known To Infect Humans ▿ †"

    Article Title: 90-Kilodalton Heat Shock Protein, Hsp90, as a Target for Genotyping Cryptosporidium spp. Known To Infect Humans ▿ spp. Known To Infect Humans ▿ †

    Journal: Eukaryotic Cell

    doi: 10.1128/EC.00294-08

    Differentiation of gastric Cryptosporidium spp. by RFLP analysis of hsp90 PCR gene products using BbsI. Lanes 1 and 11, 100-bp molecular markers; lanes 2, 3, 6, 7, and 10, C. muris ; lanes 4, 5, 8, and 9, C. andersoni .
    Figure Legend Snippet: Differentiation of gastric Cryptosporidium spp. by RFLP analysis of hsp90 PCR gene products using BbsI. Lanes 1 and 11, 100-bp molecular markers; lanes 2, 3, 6, 7, and 10, C. muris ; lanes 4, 5, 8, and 9, C. andersoni .

    Techniques Used: Polymerase Chain Reaction

    Related Articles

    Polymerase Chain Reaction:

    Article Title: 90-Kilodalton Heat Shock Protein, Hsp90, as a Target for Genotyping Cryptosporidium spp. Known To Infect Humans ▿ spp. Known To Infect Humans ▿ †
    Article Snippet: .. Ten microliters of the secondary PCR products of the Hsp90 gene was digested with StyI, HphI, or BbsI (New England BioLabs, Ipswich, MA) under the conditions suggested by the manufacturer, and the products were visualized by electrophoresis through a 2% agarose gel. ..

    Article Title: Preparation of Group I Introns for Biochemical Studies and Crystallization Assays by Native Affinity Purification
    Article Snippet: Gels were stained using ethidium bromide (Sigma #46067-50ML-F) and visualized using a UV-light transilluminator. .. Cloning procedure and transformation The PCR amplified products were purified using the QIAquick PCR purification kit (Qiagen #28104), and digested using the appropriate restriction enzymes (EcoRI, New England Biolabs (NEB) #R0101L; and NcoI, NEB #R0193L—incomplete digestion products were typically obtained using BbsI, NEB #R0539L, which made us avoid using this enzyme). .. The digested products were then purified by agarose gel electrophoresis, and eluted in a final volume of 30 µL using the QIAquick gel extraction kit (Qiagen #28706).

    Electrophoresis:

    Article Title: 90-Kilodalton Heat Shock Protein, Hsp90, as a Target for Genotyping Cryptosporidium spp. Known To Infect Humans ▿ spp. Known To Infect Humans ▿ †
    Article Snippet: .. Ten microliters of the secondary PCR products of the Hsp90 gene was digested with StyI, HphI, or BbsI (New England BioLabs, Ipswich, MA) under the conditions suggested by the manufacturer, and the products were visualized by electrophoresis through a 2% agarose gel. ..

    Agarose Gel Electrophoresis:

    Article Title: 90-Kilodalton Heat Shock Protein, Hsp90, as a Target for Genotyping Cryptosporidium spp. Known To Infect Humans ▿ spp. Known To Infect Humans ▿ †
    Article Snippet: .. Ten microliters of the secondary PCR products of the Hsp90 gene was digested with StyI, HphI, or BbsI (New England BioLabs, Ipswich, MA) under the conditions suggested by the manufacturer, and the products were visualized by electrophoresis through a 2% agarose gel. ..

    Article Title: Knock-Down of a Novel snoRNA in Tetrahymena Reveals a Dual Role in 5.8S rRNA Processing and Generation of a 26S rRNA Fragment
    Article Snippet: .. The gDNA was subsequently digested with BbsI (NEB, Ipswich, MA, USA), PCI extracted, and precipitated before 5 µg was resolved on a 0.7% agarose gel and transferred to a Hybond-N+ membrane (GE Healthcare, Chicago, IL, USA). .. The blot was analyzed by hybridization analysis using a probe made by random primer labeling of the 5′ flanking element of theTtnuCD32 mutant construct.

    Transformation Assay:

    Article Title: Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory
    Article Snippet: .. To insert the target inside pKanCiU6-1p-sgRNAscaffold, 1.5 µL of cutsmart buffer (NEB, Ipswich, MA, USA), 100 ng of pKanCiU6-1p-sgRNAscaffold, 4.2 ng of target preparation, 10 U of BbsI (NEB, Ipswich, MA, USA), 40 U of T4 DNA ligase (NEB, Ipswich, MA, USA), and deionized water up to 15 µL were mixed and the digestion/ligation reaction was performed in a thermocycler using the following parameters: 3 cycles including 37 °C for 10 min, 10 °C for 5 min and 20 °C for 5 min and then 10 cycles including 37 °C for 10 min, 10 °C for 5 min, 20 °C for 10 min. Then Escherichia coli TOP 10 thermo-competent bacteria (In Vitrogen, Waltham, MA, USA) were transformed with 5 µL of the digestion/ligation product, and selected on LB agar plate containing 25 µg/mL kanamycin and 1 mM IPTG overnight at 37 °C. ..

    Article Title: Preparation of Group I Introns for Biochemical Studies and Crystallization Assays by Native Affinity Purification
    Article Snippet: Gels were stained using ethidium bromide (Sigma #46067-50ML-F) and visualized using a UV-light transilluminator. .. Cloning procedure and transformation The PCR amplified products were purified using the QIAquick PCR purification kit (Qiagen #28104), and digested using the appropriate restriction enzymes (EcoRI, New England Biolabs (NEB) #R0101L; and NcoI, NEB #R0193L—incomplete digestion products were typically obtained using BbsI, NEB #R0539L, which made us avoid using this enzyme). .. The digested products were then purified by agarose gel electrophoresis, and eluted in a final volume of 30 µL using the QIAquick gel extraction kit (Qiagen #28706).

    Clone Assay:

    Article Title: Preparation of Group I Introns for Biochemical Studies and Crystallization Assays by Native Affinity Purification
    Article Snippet: Gels were stained using ethidium bromide (Sigma #46067-50ML-F) and visualized using a UV-light transilluminator. .. Cloning procedure and transformation The PCR amplified products were purified using the QIAquick PCR purification kit (Qiagen #28104), and digested using the appropriate restriction enzymes (EcoRI, New England Biolabs (NEB) #R0101L; and NcoI, NEB #R0193L—incomplete digestion products were typically obtained using BbsI, NEB #R0539L, which made us avoid using this enzyme). .. The digested products were then purified by agarose gel electrophoresis, and eluted in a final volume of 30 µL using the QIAquick gel extraction kit (Qiagen #28706).

    Article Title: gRNA validation for wheat genome editing with the CRISPR-Cas9 system
    Article Snippet: The reaction was heated to 95 °C for 5 min and then left at room temperature for 30 min. Annealed oligos were inserted into pCR8-U6-NCgRNA by simultaneous digestion/ligation using 1 μL annealed oligos, 50 ng pCR8-U6-NCgRNA, 1X NEBuffer 2.1, 2 units BbsI (New England Biolabs), 1X T4 DNA ligase buffer, and 0.5 units T4 DNA ligase (Invitrogen) in a total reaction volume of 10 μL. .. Cycling conditions were as follows: 37 °C for 1 h, 15 °C for 1 min, 20 °C for 10 min (2 cycles), and finally 37 °C for 1 h. Positive clones of pCR8-U6-gRNA1/2/3/4/5/6/7 were identified by diagnostic double digest with BbsI and EcoRI-HF (New England Biolabs), and validated by Sanger sequencing (Australian Genome Research Facility). .. To construct the Cas9 vector, the rice codon-optimised SpCas9 gene with N- and C-terminal nuclear localisation signals [ ] was synthesised (GenScript) and inserted into the generic vector pUbi-rbcS as an NcoI –AscI fragment between the maize Ubiquitin 1 promoter [ , ] and the wheat rbcS Class II terminator [ ], resulting in pUbi-Cas9-rbcS.

    Amplification:

    Article Title: Preparation of Group I Introns for Biochemical Studies and Crystallization Assays by Native Affinity Purification
    Article Snippet: Gels were stained using ethidium bromide (Sigma #46067-50ML-F) and visualized using a UV-light transilluminator. .. Cloning procedure and transformation The PCR amplified products were purified using the QIAquick PCR purification kit (Qiagen #28104), and digested using the appropriate restriction enzymes (EcoRI, New England Biolabs (NEB) #R0101L; and NcoI, NEB #R0193L—incomplete digestion products were typically obtained using BbsI, NEB #R0539L, which made us avoid using this enzyme). .. The digested products were then purified by agarose gel electrophoresis, and eluted in a final volume of 30 µL using the QIAquick gel extraction kit (Qiagen #28706).

    Purification:

    Article Title: Preparation of Group I Introns for Biochemical Studies and Crystallization Assays by Native Affinity Purification
    Article Snippet: Gels were stained using ethidium bromide (Sigma #46067-50ML-F) and visualized using a UV-light transilluminator. .. Cloning procedure and transformation The PCR amplified products were purified using the QIAquick PCR purification kit (Qiagen #28104), and digested using the appropriate restriction enzymes (EcoRI, New England Biolabs (NEB) #R0101L; and NcoI, NEB #R0193L—incomplete digestion products were typically obtained using BbsI, NEB #R0539L, which made us avoid using this enzyme). .. The digested products were then purified by agarose gel electrophoresis, and eluted in a final volume of 30 µL using the QIAquick gel extraction kit (Qiagen #28706).

    Diagnostic Assay:

    Article Title: gRNA validation for wheat genome editing with the CRISPR-Cas9 system
    Article Snippet: The reaction was heated to 95 °C for 5 min and then left at room temperature for 30 min. Annealed oligos were inserted into pCR8-U6-NCgRNA by simultaneous digestion/ligation using 1 μL annealed oligos, 50 ng pCR8-U6-NCgRNA, 1X NEBuffer 2.1, 2 units BbsI (New England Biolabs), 1X T4 DNA ligase buffer, and 0.5 units T4 DNA ligase (Invitrogen) in a total reaction volume of 10 μL. .. Cycling conditions were as follows: 37 °C for 1 h, 15 °C for 1 min, 20 °C for 10 min (2 cycles), and finally 37 °C for 1 h. Positive clones of pCR8-U6-gRNA1/2/3/4/5/6/7 were identified by diagnostic double digest with BbsI and EcoRI-HF (New England Biolabs), and validated by Sanger sequencing (Australian Genome Research Facility). .. To construct the Cas9 vector, the rice codon-optimised SpCas9 gene with N- and C-terminal nuclear localisation signals [ ] was synthesised (GenScript) and inserted into the generic vector pUbi-rbcS as an NcoI –AscI fragment between the maize Ubiquitin 1 promoter [ , ] and the wheat rbcS Class II terminator [ ], resulting in pUbi-Cas9-rbcS.

    Sequencing:

    Article Title: gRNA validation for wheat genome editing with the CRISPR-Cas9 system
    Article Snippet: The reaction was heated to 95 °C for 5 min and then left at room temperature for 30 min. Annealed oligos were inserted into pCR8-U6-NCgRNA by simultaneous digestion/ligation using 1 μL annealed oligos, 50 ng pCR8-U6-NCgRNA, 1X NEBuffer 2.1, 2 units BbsI (New England Biolabs), 1X T4 DNA ligase buffer, and 0.5 units T4 DNA ligase (Invitrogen) in a total reaction volume of 10 μL. .. Cycling conditions were as follows: 37 °C for 1 h, 15 °C for 1 min, 20 °C for 10 min (2 cycles), and finally 37 °C for 1 h. Positive clones of pCR8-U6-gRNA1/2/3/4/5/6/7 were identified by diagnostic double digest with BbsI and EcoRI-HF (New England Biolabs), and validated by Sanger sequencing (Australian Genome Research Facility). .. To construct the Cas9 vector, the rice codon-optimised SpCas9 gene with N- and C-terminal nuclear localisation signals [ ] was synthesised (GenScript) and inserted into the generic vector pUbi-rbcS as an NcoI –AscI fragment between the maize Ubiquitin 1 promoter [ , ] and the wheat rbcS Class II terminator [ ], resulting in pUbi-Cas9-rbcS.

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    • bbsi  (New England Biolabs)
    99
    New England Biolabs bbsi
    A general strategy to synthesize and purify group I introns within the corresponding exons. (A) General scheme of synthesis by PCR from overlapping primers followed by native purification of the transcribed RNA. The variant shown contains a group I intron (red), 5′ and 3′ exons (purple), a T7 promoter (blue), and restriction sites suitable for cloning (green). The mutated HDV, the dual SRP tag, and the Tma M protein coupled to a support are shown in orange, black, and cyan, respectively. Note that the <t>NcoI</t> restriction site was engineered within the 5′ end of the HDV mutant [3] , while the <t>BbsI</t> restriction site is no longer present in the final RNA. (B) Affinity purification chart for small-scale transcription reactions developed in this study (WB: wash buffer; CB: cleavage buffer; SB: storage buffer; RB: regeneration buffer; see Materials and Methods section).
    Bbsi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bbsi/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bbsi - by Bioz Stars, 2021-06
    99/100 stars
    		  Buy from Supplier

    Image Search Results


    A general strategy to synthesize and purify group I introns within the corresponding exons. (A) General scheme of synthesis by PCR from overlapping primers followed by native purification of the transcribed RNA. The variant shown contains a group I intron (red), 5′ and 3′ exons (purple), a T7 promoter (blue), and restriction sites suitable for cloning (green). The mutated HDV, the dual SRP tag, and the Tma M protein coupled to a support are shown in orange, black, and cyan, respectively. Note that the NcoI restriction site was engineered within the 5′ end of the HDV mutant [3] , while the BbsI restriction site is no longer present in the final RNA. (B) Affinity purification chart for small-scale transcription reactions developed in this study (WB: wash buffer; CB: cleavage buffer; SB: storage buffer; RB: regeneration buffer; see Materials and Methods section).

    Journal: PLoS ONE

    Article Title: Preparation of Group I Introns for Biochemical Studies and Crystallization Assays by Native Affinity Purification

    doi: 10.1371/journal.pone.0006740

    Figure Lengend Snippet: A general strategy to synthesize and purify group I introns within the corresponding exons. (A) General scheme of synthesis by PCR from overlapping primers followed by native purification of the transcribed RNA. The variant shown contains a group I intron (red), 5′ and 3′ exons (purple), a T7 promoter (blue), and restriction sites suitable for cloning (green). The mutated HDV, the dual SRP tag, and the Tma M protein coupled to a support are shown in orange, black, and cyan, respectively. Note that the NcoI restriction site was engineered within the 5′ end of the HDV mutant [3] , while the BbsI restriction site is no longer present in the final RNA. (B) Affinity purification chart for small-scale transcription reactions developed in this study (WB: wash buffer; CB: cleavage buffer; SB: storage buffer; RB: regeneration buffer; see Materials and Methods section).

    Article Snippet: Cloning procedure and transformation The PCR amplified products were purified using the QIAquick PCR purification kit (Qiagen #28104), and digested using the appropriate restriction enzymes (EcoRI, New England Biolabs (NEB) #R0101L; and NcoI, NEB #R0193L—incomplete digestion products were typically obtained using BbsI, NEB #R0539L, which made us avoid using this enzyme).

    Techniques: Polymerase Chain Reaction, Purification, Variant Assay, Clone Assay, Mutagenesis, Affinity Purification, Western Blot

    Architecture of the MCS-IIS C2. This DNA-sequence is located on the cargo vector between the E. coli ori and the Bacillus antibiotic marker. The recognition sites for five type IIS restriction enzymes (AarI, BtgZI, BbsI, BsaI, BsmBI), each designed to create a 5′ GCGA-overhang are encoded on the DNA stretch. Architecture of all MCS-IIS can be found in Fig. S1 .

    Journal: Scientific Reports

    Article Title: Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis

    doi: 10.1038/s41598-017-14329-5

    Figure Lengend Snippet: Architecture of the MCS-IIS C2. This DNA-sequence is located on the cargo vector between the E. coli ori and the Bacillus antibiotic marker. The recognition sites for five type IIS restriction enzymes (AarI, BtgZI, BbsI, BsaI, BsmBI), each designed to create a 5′ GCGA-overhang are encoded on the DNA stretch. Architecture of all MCS-IIS can be found in Fig. S1 .

    Article Snippet: BsaI, BbsI, BsmBI and BtgZI were purchased from NEB, AarI from Thermo ScientificTM .

    Techniques: Sequencing, Plasmid Preparation, Marker

    Overview of the experimental design for CiPDS disruption. ( A ) Schematic position of the two guide RNAs (red boxes) targeting the fifth exon of CiPDS . The blue boxes indicate exons; the black lines indicate introns. ( B ) Schematic view of pKanCiU6-1p-sgRNAscaffold. Between the CiU6-1p and the sgRNA scaffold (scaff), a ccdB gene driven by the LacZ promoter (counter selection marker) is surrounded by BbsI (second-generation enzyme) recognition sites, which allows the ligation of the hybridized guide adaptor shown in C . Red B-L depicts the orientation of the BbsI recognition site which achieves the cleavage on the left side. Blue B-R depicts the orientation of the BbsI recognition site which achieves cleavage on the right side. ( C ) Sequences of the complementary guide adaptors with the 19 pb guide sequence (yellow), the transcription initiator nucleotide G (bold), and the binding sites necessary to insert the hybridized guide adaptor into the pKanCiU6-1p-sgRNAscaffold (red). ( D ) Representation of the vector resulting from the insertion of the hybridized guide adaptors in the pKanCiU6-1p-sgRNAscaffold, (note that one vector is constructed for each guide). The primers used for preparation of CiU6-1p-guide-sgRNA cassettes for Golden Gate Cloning are also shown. ( E ) Representation of the final plasmid pYLCRISPR-sgRNA1-sgRNA2 resulting from the cloning of the two cassettes into the pYLCRISPR/Cas9P 35S -B [ 36 ]. RB = Right Border, LB = Left Border, NLS = Nuclear Localization Signal.

    Journal: International Journal of Molecular Sciences

    Article Title: Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory

    doi: 10.3390/ijms20051155

    Figure Lengend Snippet: Overview of the experimental design for CiPDS disruption. ( A ) Schematic position of the two guide RNAs (red boxes) targeting the fifth exon of CiPDS . The blue boxes indicate exons; the black lines indicate introns. ( B ) Schematic view of pKanCiU6-1p-sgRNAscaffold. Between the CiU6-1p and the sgRNA scaffold (scaff), a ccdB gene driven by the LacZ promoter (counter selection marker) is surrounded by BbsI (second-generation enzyme) recognition sites, which allows the ligation of the hybridized guide adaptor shown in C . Red B-L depicts the orientation of the BbsI recognition site which achieves the cleavage on the left side. Blue B-R depicts the orientation of the BbsI recognition site which achieves cleavage on the right side. ( C ) Sequences of the complementary guide adaptors with the 19 pb guide sequence (yellow), the transcription initiator nucleotide G (bold), and the binding sites necessary to insert the hybridized guide adaptor into the pKanCiU6-1p-sgRNAscaffold (red). ( D ) Representation of the vector resulting from the insertion of the hybridized guide adaptors in the pKanCiU6-1p-sgRNAscaffold, (note that one vector is constructed for each guide). The primers used for preparation of CiU6-1p-guide-sgRNA cassettes for Golden Gate Cloning are also shown. ( E ) Representation of the final plasmid pYLCRISPR-sgRNA1-sgRNA2 resulting from the cloning of the two cassettes into the pYLCRISPR/Cas9P 35S -B [ 36 ]. RB = Right Border, LB = Left Border, NLS = Nuclear Localization Signal.

    Article Snippet: To insert the target inside pKanCiU6-1p-sgRNAscaffold, 1.5 µL of cutsmart buffer (NEB, Ipswich, MA, USA), 100 ng of pKanCiU6-1p-sgRNAscaffold, 4.2 ng of target preparation, 10 U of BbsI (NEB, Ipswich, MA, USA), 40 U of T4 DNA ligase (NEB, Ipswich, MA, USA), and deionized water up to 15 µL were mixed and the digestion/ligation reaction was performed in a thermocycler using the following parameters: 3 cycles including 37 °C for 10 min, 10 °C for 5 min and 20 °C for 5 min and then 10 cycles including 37 °C for 10 min, 10 °C for 5 min, 20 °C for 10 min. Then Escherichia coli TOP 10 thermo-competent bacteria (In Vitrogen, Waltham, MA, USA) were transformed with 5 µL of the digestion/ligation product, and selected on LB agar plate containing 25 µg/mL kanamycin and 1 mM IPTG overnight at 37 °C.

    Techniques: Selection, Marker, Ligation, Sequencing, Binding Assay, Plasmid Preparation, Construct, Clone Assay