bbsi  (New England Biolabs)


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    BbsI 1 500 units
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    Restriction Enzymes
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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 1432 article reviews
    Price from $9.99 to $1999.99
    bbsi - by Bioz Stars, 2020-09
    99/100 stars

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    Images

    1) 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

    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 "Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments"

    Article Title: Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments

    Journal: Scientific Reports

    doi: 10.1038/srep10655

    Lambda phage genome construction. ( A ) Design of the OGAB blocks. A total of 48.5 kb in length of lambda phage genome was divided into 50 fragments, each of which was cloned into cloning vector pMD19. Three restriction enzyme sites, BbsI (green), AarI (red), and BsmBI (blue), at least one of which did not appear in each OGAB block, were used. ( B ) The 50 OGAB block mixtures before (blue) and after (red) size selection by electrophoresis were compared relative to the population by quantitative PCR. The apparent CV mol values for the OGAB blocks before and after size selection were 7.4% and 7.0%; however, due to the 3.6% measurement error of the PCR machine, the error-corrected CV mol values were 7.0% and 6.6%, respectively. The population profile after size selection was almost the same as that before size selection. We performed two additional OGAB block preparations using independently measured and mixed OGAB block plasmids and determined CV mol (%), resulting in a similar value to that of the initial experiment (CV mol (%)(error-corrected) = 6.8% and 7.3%). All raw data are indicated in Supplemental Table S8 . ( C ) Restriction digestion pattern of plasmids from 12 randomly selected transformants. HindIII and SfiI were used for double digestion. In the case of four clones (numbers circled), except for the 15 kb of the assembly vector pGET118-AarI, all of the bands were the same as the commercial size marker λ/HindIII. ( D ) Plaque formation assay of correctly assembled plasmid. The four plasmids were digested with lambda terminase, packed into lambda phage extract, and used to infect E. coli . There were no differences in features between the assembled plasmid-born plaque and the authentic lambda phage DNA-born plaque. ( E ) Confirmation of the plaque as designed. Due to an intended synonymous codon mutation in OGAB block 10, a restriction enzyme AvaI site appeared at the largest fragment (14,678 bp) of the wild type, generating two fragments (9,885 and 4,793 bp). All of the clones had restriction patterns at most large AvaI fragments distinct from those of the wild type, indicating that these phages originated from assembled DNAs.
    Figure Legend Snippet: Lambda phage genome construction. ( A ) Design of the OGAB blocks. A total of 48.5 kb in length of lambda phage genome was divided into 50 fragments, each of which was cloned into cloning vector pMD19. Three restriction enzyme sites, BbsI (green), AarI (red), and BsmBI (blue), at least one of which did not appear in each OGAB block, were used. ( B ) The 50 OGAB block mixtures before (blue) and after (red) size selection by electrophoresis were compared relative to the population by quantitative PCR. The apparent CV mol values for the OGAB blocks before and after size selection were 7.4% and 7.0%; however, due to the 3.6% measurement error of the PCR machine, the error-corrected CV mol values were 7.0% and 6.6%, respectively. The population profile after size selection was almost the same as that before size selection. We performed two additional OGAB block preparations using independently measured and mixed OGAB block plasmids and determined CV mol (%), resulting in a similar value to that of the initial experiment (CV mol (%)(error-corrected) = 6.8% and 7.3%). All raw data are indicated in Supplemental Table S8 . ( C ) Restriction digestion pattern of plasmids from 12 randomly selected transformants. HindIII and SfiI were used for double digestion. In the case of four clones (numbers circled), except for the 15 kb of the assembly vector pGET118-AarI, all of the bands were the same as the commercial size marker λ/HindIII. ( D ) Plaque formation assay of correctly assembled plasmid. The four plasmids were digested with lambda terminase, packed into lambda phage extract, and used to infect E. coli . There were no differences in features between the assembled plasmid-born plaque and the authentic lambda phage DNA-born plaque. ( E ) Confirmation of the plaque as designed. Due to an intended synonymous codon mutation in OGAB block 10, a restriction enzyme AvaI site appeared at the largest fragment (14,678 bp) of the wild type, generating two fragments (9,885 and 4,793 bp). All of the clones had restriction patterns at most large AvaI fragments distinct from those of the wild type, indicating that these phages originated from assembled DNAs.

    Techniques Used: Clone Assay, Plasmid Preparation, Blocking Assay, Selection, Electrophoresis, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Marker, Plaque Formation Assay, Mutagenesis

    4) 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

    5) 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

    6) 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

    Related Articles

    Clone Assay:

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

    Amplification:

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

    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. ..

    Purification:

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

    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: .. 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).

    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: .. 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).

    Plasmid Preparation:

    Article Title: Rapid generation of gene-targeted EPS-derived mouse models through tetraploid complementation
    Article Snippet: .. The pX330 vector was digested by BbsI (NEB) for 16 h at 37 °C. .. The IL3 or IL6 gRNA-pX330 vector was constructed by ligating the gRNA annealed product and the pX330 digested product.

    Article Title: Genetic Resistance to Avian Leukosis Viruses Induced by CRISPR/Cas9 Editing of Specific Receptor Genes in Chicken Cells
    Article Snippet: .. The PX458 vector was cleaved by BbsI and annealed oligonucleotides were ligated by Quick ligase (New England Biolabs) to form pX458-TVA, pX458TVC, and pX458-TVJ1/2/4. .. The chicken cell line DF-1 [ ] was grown in a mixture of two parts Dulbecco’s modified Eagle’s medium and 1 part F-12 medium supplemented with 8% fetal calf serum, 2% chicken serum, and 1× antibiotic-antimycotic solution (Sigma, St. Louis, MO, USA) under 5% CO2 atmosphere at 37 °C.

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    • bbsi  (New England Biolabs)
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    New England Biolabs bbsi
    Differentiation of gastric Cryptosporidium spp. by RFLP analysis of hsp90 <t>PCR</t> gene products using <t>BbsI.</t> 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 .
    Bbsi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 71 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 71 article reviews
    Price from $9.99 to $1999.99
    bbsi - by Bioz Stars, 2020-09
    99/100 stars
    		  Buy from Supplier

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    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 .

    Journal: Eukaryotic Cell

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

    doi: 10.1128/EC.00294-08

    Figure Lengend 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 .

    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.

    Techniques: Polymerase Chain Reaction

    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

    Lambda phage genome construction. ( A ) Design of the OGAB blocks. A total of 48.5 kb in length of lambda phage genome was divided into 50 fragments, each of which was cloned into cloning vector pMD19. Three restriction enzyme sites, BbsI (green), AarI (red), and BsmBI (blue), at least one of which did not appear in each OGAB block, were used. ( B ) The 50 OGAB block mixtures before (blue) and after (red) size selection by electrophoresis were compared relative to the population by quantitative PCR. The apparent CV mol values for the OGAB blocks before and after size selection were 7.4% and 7.0%; however, due to the 3.6% measurement error of the PCR machine, the error-corrected CV mol values were 7.0% and 6.6%, respectively. The population profile after size selection was almost the same as that before size selection. We performed two additional OGAB block preparations using independently measured and mixed OGAB block plasmids and determined CV mol (%), resulting in a similar value to that of the initial experiment (CV mol (%)(error-corrected) = 6.8% and 7.3%). All raw data are indicated in Supplemental Table S8 . ( C ) Restriction digestion pattern of plasmids from 12 randomly selected transformants. HindIII and SfiI were used for double digestion. In the case of four clones (numbers circled), except for the 15 kb of the assembly vector pGET118-AarI, all of the bands were the same as the commercial size marker λ/HindIII. ( D ) Plaque formation assay of correctly assembled plasmid. The four plasmids were digested with lambda terminase, packed into lambda phage extract, and used to infect E. coli . There were no differences in features between the assembled plasmid-born plaque and the authentic lambda phage DNA-born plaque. ( E ) Confirmation of the plaque as designed. Due to an intended synonymous codon mutation in OGAB block 10, a restriction enzyme AvaI site appeared at the largest fragment (14,678 bp) of the wild type, generating two fragments (9,885 and 4,793 bp). All of the clones had restriction patterns at most large AvaI fragments distinct from those of the wild type, indicating that these phages originated from assembled DNAs.

    Journal: Scientific Reports

    Article Title: Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments

    doi: 10.1038/srep10655

    Figure Lengend Snippet: Lambda phage genome construction. ( A ) Design of the OGAB blocks. A total of 48.5 kb in length of lambda phage genome was divided into 50 fragments, each of which was cloned into cloning vector pMD19. Three restriction enzyme sites, BbsI (green), AarI (red), and BsmBI (blue), at least one of which did not appear in each OGAB block, were used. ( B ) The 50 OGAB block mixtures before (blue) and after (red) size selection by electrophoresis were compared relative to the population by quantitative PCR. The apparent CV mol values for the OGAB blocks before and after size selection were 7.4% and 7.0%; however, due to the 3.6% measurement error of the PCR machine, the error-corrected CV mol values were 7.0% and 6.6%, respectively. The population profile after size selection was almost the same as that before size selection. We performed two additional OGAB block preparations using independently measured and mixed OGAB block plasmids and determined CV mol (%), resulting in a similar value to that of the initial experiment (CV mol (%)(error-corrected) = 6.8% and 7.3%). All raw data are indicated in Supplemental Table S8 . ( C ) Restriction digestion pattern of plasmids from 12 randomly selected transformants. HindIII and SfiI were used for double digestion. In the case of four clones (numbers circled), except for the 15 kb of the assembly vector pGET118-AarI, all of the bands were the same as the commercial size marker λ/HindIII. ( D ) Plaque formation assay of correctly assembled plasmid. The four plasmids were digested with lambda terminase, packed into lambda phage extract, and used to infect E. coli . There were no differences in features between the assembled plasmid-born plaque and the authentic lambda phage DNA-born plaque. ( E ) Confirmation of the plaque as designed. Due to an intended synonymous codon mutation in OGAB block 10, a restriction enzyme AvaI site appeared at the largest fragment (14,678 bp) of the wild type, generating two fragments (9,885 and 4,793 bp). All of the clones had restriction patterns at most large AvaI fragments distinct from those of the wild type, indicating that these phages originated from assembled DNAs.

    Article Snippet: Restriction endonucleases BbsI and BsmBI were purchased from NEB.

    Techniques: Clone Assay, Plasmid Preparation, Blocking Assay, Selection, Electrophoresis, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Marker, Plaque Formation Assay, Mutagenesis