bsg i  (New England Biolabs)


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  • 97
    Name:
    BsgI
    Description:
    BsgI 250 units
    Catalog Number:
    r0559l
    Price:
    282
    Size:
    250 units
    Category:
    Restriction Enzymes
    Buy from Supplier


    Structured Review

    New England Biolabs bsg i
    BsgI
    BsgI 250 units
    https://www.bioz.com/result/bsg i/product/New England Biolabs
    Average 97 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    bsg i - by Bioz Stars, 2020-09
    97/100 stars

    Images

    1) Product Images from "A novel c.-22T > C mutation in GALK1 promoter is associated with elevated galactokinase phenotype"

    Article Title: A novel c.-22T > C mutation in GALK1 promoter is associated with elevated galactokinase phenotype

    Journal: BMC Medical Genetics

    doi: 10.1186/1471-2350-10-29

    Identification of sequence variations in the GALK1 promoter region . Sequencing revealed the presence of a mutation of c.-22T > C (A). Gel electrophoresis patterns of PCR amplified DNA fragments digested with Bsg I for the confirmation of c.-22T > C (B) are shown (Lane M, DNA size marker; lane 1, C/C; lane2, T/C; lane 3, T/T). Bsg I recognizes the sequence GTGCAG (underlined in A electrophoretogram) present in wild type c.-22T, but not in c.-22C. The PCR fragment from genotype c.-22C/C, c.-22T/C and c.-22T/T will produce (à produce) one (339 bp), three (339, 267 and 72 bp) and two (267 and 72 bp) bands.
    Figure Legend Snippet: Identification of sequence variations in the GALK1 promoter region . Sequencing revealed the presence of a mutation of c.-22T > C (A). Gel electrophoresis patterns of PCR amplified DNA fragments digested with Bsg I for the confirmation of c.-22T > C (B) are shown (Lane M, DNA size marker; lane 1, C/C; lane2, T/C; lane 3, T/T). Bsg I recognizes the sequence GTGCAG (underlined in A electrophoretogram) present in wild type c.-22T, but not in c.-22C. The PCR fragment from genotype c.-22C/C, c.-22T/C and c.-22T/T will produce (à produce) one (339 bp), three (339, 267 and 72 bp) and two (267 and 72 bp) bands.

    Techniques Used: Sequencing, Mutagenesis, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Amplification, Marker

    2) Product Images from "High-Throughput Genotyping of Green Algal Mutants Reveals Random Distribution of Mutagenic Insertion Sites and Endonucleolytic Cleavage of Transforming DNA [W]High-Throughput Genotyping of Green Algal Mutants Reveals Random Distribution of Mutagenic Insertion Sites and Endonucleolytic Cleavage of Transforming DNA [W] [OPEN]"

    Article Title: High-Throughput Genotyping of Green Algal Mutants Reveals Random Distribution of Mutagenic Insertion Sites and Endonucleolytic Cleavage of Transforming DNA [W]High-Throughput Genotyping of Green Algal Mutants Reveals Random Distribution of Mutagenic Insertion Sites and Endonucleolytic Cleavage of Transforming DNA [W] [OPEN]

    Journal: The Plant Cell

    doi: 10.1105/tpc.114.124099

    ChlaMmeSeq Is an Mme I-Based Strategy for Mapping Insertion Sites. (A) The cassette used to transform C. reinhardtii cells is 2660 bp long and is composed of a PSAD promoter, AphVIII gene (conferring paromomycin resistance), CYTc6 intron, and PSAD and RPL12 terminators in opposite orientations. Restriction enzyme sites ( Mme I and Bsg I) are shown. Blue lines represent genomic DNA. (B) Double digestion of mutant genomic DNA with Mme I and Bsg I yields a 1121-bp fragment from each side of the cassette, containing 20 to 21 bp of flanking genomic DNA with a two-nucleotide overhang. (C) An adaptor (orange) that contains both PCR and sequencing primer binding sites is ligated to the digestion products. (D) The flanking DNA sequences are amplified with PCR primers (black and orange arrows) binding to the cassette and adaptors. (E) The resulting PCR product is sequenced by Sanger or Illumina sequencing (for individual or pooled mutants, respectively). (F) The 20- to 21-bp flanking DNA sequences are mapped to the C. reinhardtii genome to identify the site of each insertion.
    Figure Legend Snippet: ChlaMmeSeq Is an Mme I-Based Strategy for Mapping Insertion Sites. (A) The cassette used to transform C. reinhardtii cells is 2660 bp long and is composed of a PSAD promoter, AphVIII gene (conferring paromomycin resistance), CYTc6 intron, and PSAD and RPL12 terminators in opposite orientations. Restriction enzyme sites ( Mme I and Bsg I) are shown. Blue lines represent genomic DNA. (B) Double digestion of mutant genomic DNA with Mme I and Bsg I yields a 1121-bp fragment from each side of the cassette, containing 20 to 21 bp of flanking genomic DNA with a two-nucleotide overhang. (C) An adaptor (orange) that contains both PCR and sequencing primer binding sites is ligated to the digestion products. (D) The flanking DNA sequences are amplified with PCR primers (black and orange arrows) binding to the cassette and adaptors. (E) The resulting PCR product is sequenced by Sanger or Illumina sequencing (for individual or pooled mutants, respectively). (F) The 20- to 21-bp flanking DNA sequences are mapped to the C. reinhardtii genome to identify the site of each insertion.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Sequencing, Binding Assay, Amplification

    3) Product Images from "DNA looping by two-site restriction endonucleases: heterogeneous probability distributions for loop size and unbinding force"

    Article Title: DNA looping by two-site restriction endonucleases: heterogeneous probability distributions for loop size and unbinding force

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl382

    Typical DNA force-extension plots. ( A ) The two-site enzyme BsgI tested on the LBAC-B template, which has four recognition sites. The one detected loop had a measured length of 946 bp, in excellent agreement with the separation between the binding sites at positions 8342 and 9287 bp (a distance of 945 bp). ( B ) Sau3AI on the LBAC-A template, which has 55 recognition sites. ( C ) Control experiments: (i) the two-site enzyme Sau3AI was tested on bacteriophage phiX174 DNA, which contains no copies of its recognition sequence; (ii) the one-site enzyme HaeIII was tested on LBAC-B DNA containing 36 copies of its recognition sequence; (iii) the two-site enzyme SfiI was tested on LBAC-B DNA containing one copy of its recognition sequence and (iv) LBAC-B DNA, containing 26 FokI binding sites, incubated in the FokI reaction buffer, but no FokI added.
    Figure Legend Snippet: Typical DNA force-extension plots. ( A ) The two-site enzyme BsgI tested on the LBAC-B template, which has four recognition sites. The one detected loop had a measured length of 946 bp, in excellent agreement with the separation between the binding sites at positions 8342 and 9287 bp (a distance of 945 bp). ( B ) Sau3AI on the LBAC-A template, which has 55 recognition sites. ( C ) Control experiments: (i) the two-site enzyme Sau3AI was tested on bacteriophage phiX174 DNA, which contains no copies of its recognition sequence; (ii) the one-site enzyme HaeIII was tested on LBAC-B DNA containing 36 copies of its recognition sequence; (iii) the two-site enzyme SfiI was tested on LBAC-B DNA containing one copy of its recognition sequence and (iv) LBAC-B DNA, containing 26 FokI binding sites, incubated in the FokI reaction buffer, but no FokI added.

    Techniques Used: Binding Assay, Sequencing, Incubation

    4) Product Images from "Molecular Cloning and Mutagenesis of a DNA Locus Involved in Lipooligosaccharide Biosynthesis in Haemophilus somnus"

    Article Title: Molecular Cloning and Mutagenesis of a DNA Locus Involved in Lipooligosaccharide Biosynthesis in Haemophilus somnus

    Journal: Infection and Immunity

    doi:

    Restriction endonuclease map and construction of plasmids used to make allelic exchange mutants and to complement LOS genes in H. somnus ). pCAAT was digested with Eco RI to obtain the 3.9-kb fragment, and pGEM-3Z was digested with Hin cII to obtain a 2.7-kb fragment. These fragments were ligated together to obtain 6.6-kb plasmid pCAAT-SalI, which was further digested with Sal I- Bsg I to obtain a 6.1-kb fragment with a 630-bp deletion in lob-2A . The 1.2-kb Sma I fragment from pUC4-kixx containing the Tn 5 Km r cassette was then ligated into the Sal I- Bsg I site of pCAAT-SalI to obtain 7.3-kb plasmid pCAATΔlob2A. The 8.8-kb plasmid pLSGA was constructed by digesting pCAAT with Eco RI and cloning the 3.9-kb fragment into the Sma I site of pLS88. pLSGA was then cut with Xba I and Hpa I to remove part of lob-1 and the non-LOS afu -like genes, and the 6.84-kb plasmid was ligated to itself. All ligations were done following blunt ending of the plasmids.
    Figure Legend Snippet: Restriction endonuclease map and construction of plasmids used to make allelic exchange mutants and to complement LOS genes in H. somnus ). pCAAT was digested with Eco RI to obtain the 3.9-kb fragment, and pGEM-3Z was digested with Hin cII to obtain a 2.7-kb fragment. These fragments were ligated together to obtain 6.6-kb plasmid pCAAT-SalI, which was further digested with Sal I- Bsg I to obtain a 6.1-kb fragment with a 630-bp deletion in lob-2A . The 1.2-kb Sma I fragment from pUC4-kixx containing the Tn 5 Km r cassette was then ligated into the Sal I- Bsg I site of pCAAT-SalI to obtain 7.3-kb plasmid pCAATΔlob2A. The 8.8-kb plasmid pLSGA was constructed by digesting pCAAT with Eco RI and cloning the 3.9-kb fragment into the Sma I site of pLS88. pLSGA was then cut with Xba I and Hpa I to remove part of lob-1 and the non-LOS afu -like genes, and the 6.84-kb plasmid was ligated to itself. All ligations were done following blunt ending of the plasmids.

    Techniques Used: Plasmid Preparation, Construct, Clone Assay

    Related Articles

    Mutagenesis:

    Article Title: A novel c.-22T > C mutation in GALK1 promoter is associated with elevated galactokinase phenotype
    Article Snippet: .. Bsg I (New England Biolabs, MA, USA) restriction enzyme was used to confirm the c-22T > C mutation on the PCR products, using the primer pair, GKP2-F and GKP2-R, which was the same as used for sequencing. .. However, Pvu II (New England Biolabs, MA, USA) GKP2-R' (5'-CGT GCA GCC CCT CAC CAT AG-3') primer was used to confirm the c.-179A > G and c.-27A > C mutations.

    Construct:

    Article Title: A Role for Nonessential Domain II of Initiator Protein, DnaA, in Replication Control
    Article Snippet: .. The resulting construct, pSTL378, causes loss of a Bsg I site, which was confirmed by restriction digest using Bsg I (New England Biolabs). .. Plasmids expressing DiaA as a biotin-binding domain fusion protein were constructed using the site-specific recombination of Gateway Cloning Technology (Invitrogen).

    Purification:

    Article Title: Analysis of the transcriptome of Panax notoginseng root uncovers putative triterpene saponin-biosynthetic genes and genetic markers
    Article Snippet: .. Then the purified cDNA was treated with Bsg I (NEB, USA) overnight at 37°C and recovered by QIAquick PCR Purification Kit (Qiagen, USA). ..

    Polymerase Chain Reaction:

    Article Title: A novel c.-22T > C mutation in GALK1 promoter is associated with elevated galactokinase phenotype
    Article Snippet: .. Bsg I (New England Biolabs, MA, USA) restriction enzyme was used to confirm the c-22T > C mutation on the PCR products, using the primer pair, GKP2-F and GKP2-R, which was the same as used for sequencing. .. However, Pvu II (New England Biolabs, MA, USA) GKP2-R' (5'-CGT GCA GCC CCT CAC CAT AG-3') primer was used to confirm the c.-179A > G and c.-27A > C mutations.

    Article Title: Analysis of the transcriptome of Panax notoginseng root uncovers putative triterpene saponin-biosynthetic genes and genetic markers
    Article Snippet: .. Then the purified cDNA was treated with Bsg I (NEB, USA) overnight at 37°C and recovered by QIAquick PCR Purification Kit (Qiagen, USA). ..

    Generated:

    Article Title: T-Cell Epitopes and Human Leukocyte Antigen Restriction Elements of an Immunodominant Antigen of Blastomyces dermatitidis
    Article Snippet: .. The N terminus coding sequence (538 bp) was generated by restricting the WI-1 (strain 26199) genomic sequence with Bsg I and Kpn I (New England Biolabs). .. Bsg I cuts 51 bp (encoding 17 amino acids) 5′ of the tandem repeat coding sequence; Kpn I cuts 3′ of the WI-1 stop codon, thereby “dropping out” from the plasmid all WI-1 coding sequences except for that for the N terminus.

    other:

    Article Title: DNA looping by two-site restriction endonucleases: heterogeneous probability distributions for loop size and unbinding force
    Article Snippet: Endonucleases BamHI, BpmI, BsgI, BspMI, BstNI, EcoRI, EcoRV, FokI, HaeIII, HpaII, MboII, MspI, NarI, NaeI, SacII, Sau3AI, SfiI and SgrAI were obtained from New England Biolabs (NEB).

    Sequencing:

    Article Title: A novel c.-22T > C mutation in GALK1 promoter is associated with elevated galactokinase phenotype
    Article Snippet: .. Bsg I (New England Biolabs, MA, USA) restriction enzyme was used to confirm the c-22T > C mutation on the PCR products, using the primer pair, GKP2-F and GKP2-R, which was the same as used for sequencing. .. However, Pvu II (New England Biolabs, MA, USA) GKP2-R' (5'-CGT GCA GCC CCT CAC CAT AG-3') primer was used to confirm the c.-179A > G and c.-27A > C mutations.

    Article Title: T-Cell Epitopes and Human Leukocyte Antigen Restriction Elements of an Immunodominant Antigen of Blastomyces dermatitidis
    Article Snippet: .. The N terminus coding sequence (538 bp) was generated by restricting the WI-1 (strain 26199) genomic sequence with Bsg I and Kpn I (New England Biolabs). .. Bsg I cuts 51 bp (encoding 17 amino acids) 5′ of the tandem repeat coding sequence; Kpn I cuts 3′ of the WI-1 stop codon, thereby “dropping out” from the plasmid all WI-1 coding sequences except for that for the N terminus.

    Transformation Assay:

    Article Title: ABG1, a Novel and Essential Candida albicans Gene Encoding a Vacuolar Protein Involved in Cytokinesis and Hyphal Branching
    Article Snippet: .. Before transformation into ABG1 heterozygous strain ( abg1 Δ:: hisG/ABG1 ), pMD was linearized with BsgI (New England Biolabs). .. Plasmid pAG- ADH - ABG1 -GFP for expression of GFP-tagged Abg1p from the constitutive promoter ADH1 was created by amplifying ABG1 ORF from CAI4 genomic DNA with primers FP4 and RGFP (Table ) and ligated into the ADH-GFP plasmid (pAG1) at BamHI and SmaI sites.

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  • 97
    New England Biolabs bsg i
    Identification of sequence variations in the GALK1 promoter region . Sequencing revealed the presence of a mutation of c.-22T > C (A). Gel electrophoresis patterns of PCR amplified DNA fragments digested with <t>Bsg</t> I for the confirmation of c.-22T > C (B) are shown (Lane M, DNA size marker; lane 1, C/C; lane2, T/C; lane 3, T/T). Bsg I recognizes the sequence GTGCAG (underlined in A electrophoretogram) present in wild type c.-22T, but not in c.-22C. The PCR fragment from genotype c.-22C/C, c.-22T/C and c.-22T/T will produce (à produce) one (339 bp), three (339, 267 and 72 bp) and two (267 and 72 bp) bands.
    Bsg I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsg i/product/New England Biolabs
    Average 97 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    bsg i - by Bioz Stars, 2020-09
    97/100 stars
      Buy from Supplier

    Image Search Results


    Identification of sequence variations in the GALK1 promoter region . Sequencing revealed the presence of a mutation of c.-22T > C (A). Gel electrophoresis patterns of PCR amplified DNA fragments digested with Bsg I for the confirmation of c.-22T > C (B) are shown (Lane M, DNA size marker; lane 1, C/C; lane2, T/C; lane 3, T/T). Bsg I recognizes the sequence GTGCAG (underlined in A electrophoretogram) present in wild type c.-22T, but not in c.-22C. The PCR fragment from genotype c.-22C/C, c.-22T/C and c.-22T/T will produce (à produce) one (339 bp), three (339, 267 and 72 bp) and two (267 and 72 bp) bands.

    Journal: BMC Medical Genetics

    Article Title: A novel c.-22T > C mutation in GALK1 promoter is associated with elevated galactokinase phenotype

    doi: 10.1186/1471-2350-10-29

    Figure Lengend Snippet: Identification of sequence variations in the GALK1 promoter region . Sequencing revealed the presence of a mutation of c.-22T > C (A). Gel electrophoresis patterns of PCR amplified DNA fragments digested with Bsg I for the confirmation of c.-22T > C (B) are shown (Lane M, DNA size marker; lane 1, C/C; lane2, T/C; lane 3, T/T). Bsg I recognizes the sequence GTGCAG (underlined in A electrophoretogram) present in wild type c.-22T, but not in c.-22C. The PCR fragment from genotype c.-22C/C, c.-22T/C and c.-22T/T will produce (à produce) one (339 bp), three (339, 267 and 72 bp) and two (267 and 72 bp) bands.

    Article Snippet: Bsg I (New England Biolabs, MA, USA) restriction enzyme was used to confirm the c-22T > C mutation on the PCR products, using the primer pair, GKP2-F and GKP2-R, which was the same as used for sequencing.

    Techniques: Sequencing, Mutagenesis, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Amplification, Marker

    ChlaMmeSeq Is an Mme I-Based Strategy for Mapping Insertion Sites. (A) The cassette used to transform C. reinhardtii cells is 2660 bp long and is composed of a PSAD promoter, AphVIII gene (conferring paromomycin resistance), CYTc6 intron, and PSAD and RPL12 terminators in opposite orientations. Restriction enzyme sites ( Mme I and Bsg I) are shown. Blue lines represent genomic DNA. (B) Double digestion of mutant genomic DNA with Mme I and Bsg I yields a 1121-bp fragment from each side of the cassette, containing 20 to 21 bp of flanking genomic DNA with a two-nucleotide overhang. (C) An adaptor (orange) that contains both PCR and sequencing primer binding sites is ligated to the digestion products. (D) The flanking DNA sequences are amplified with PCR primers (black and orange arrows) binding to the cassette and adaptors. (E) The resulting PCR product is sequenced by Sanger or Illumina sequencing (for individual or pooled mutants, respectively). (F) The 20- to 21-bp flanking DNA sequences are mapped to the C. reinhardtii genome to identify the site of each insertion.

    Journal: The Plant Cell

    Article Title: High-Throughput Genotyping of Green Algal Mutants Reveals Random Distribution of Mutagenic Insertion Sites and Endonucleolytic Cleavage of Transforming DNA [W]High-Throughput Genotyping of Green Algal Mutants Reveals Random Distribution of Mutagenic Insertion Sites and Endonucleolytic Cleavage of Transforming DNA [W] [OPEN]

    doi: 10.1105/tpc.114.124099

    Figure Lengend Snippet: ChlaMmeSeq Is an Mme I-Based Strategy for Mapping Insertion Sites. (A) The cassette used to transform C. reinhardtii cells is 2660 bp long and is composed of a PSAD promoter, AphVIII gene (conferring paromomycin resistance), CYTc6 intron, and PSAD and RPL12 terminators in opposite orientations. Restriction enzyme sites ( Mme I and Bsg I) are shown. Blue lines represent genomic DNA. (B) Double digestion of mutant genomic DNA with Mme I and Bsg I yields a 1121-bp fragment from each side of the cassette, containing 20 to 21 bp of flanking genomic DNA with a two-nucleotide overhang. (C) An adaptor (orange) that contains both PCR and sequencing primer binding sites is ligated to the digestion products. (D) The flanking DNA sequences are amplified with PCR primers (black and orange arrows) binding to the cassette and adaptors. (E) The resulting PCR product is sequenced by Sanger or Illumina sequencing (for individual or pooled mutants, respectively). (F) The 20- to 21-bp flanking DNA sequences are mapped to the C. reinhardtii genome to identify the site of each insertion.

    Article Snippet: The 500-μL reactions were assembled with 3.5 μg DNA, 50 μL 10× NEB4 buffer, 1.25 μL 32 mM S -adenosyl methionine, 5 μL 10 mg/mL BSA, 40 μL 2 units/μL Mme I, and 1.25 μL 5 units/μL Bsg I (NEB).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Sequencing, Binding Assay, Amplification

    Typical DNA force-extension plots. ( A ) The two-site enzyme BsgI tested on the LBAC-B template, which has four recognition sites. The one detected loop had a measured length of 946 bp, in excellent agreement with the separation between the binding sites at positions 8342 and 9287 bp (a distance of 945 bp). ( B ) Sau3AI on the LBAC-A template, which has 55 recognition sites. ( C ) Control experiments: (i) the two-site enzyme Sau3AI was tested on bacteriophage phiX174 DNA, which contains no copies of its recognition sequence; (ii) the one-site enzyme HaeIII was tested on LBAC-B DNA containing 36 copies of its recognition sequence; (iii) the two-site enzyme SfiI was tested on LBAC-B DNA containing one copy of its recognition sequence and (iv) LBAC-B DNA, containing 26 FokI binding sites, incubated in the FokI reaction buffer, but no FokI added.

    Journal: Nucleic Acids Research

    Article Title: DNA looping by two-site restriction endonucleases: heterogeneous probability distributions for loop size and unbinding force

    doi: 10.1093/nar/gkl382

    Figure Lengend Snippet: Typical DNA force-extension plots. ( A ) The two-site enzyme BsgI tested on the LBAC-B template, which has four recognition sites. The one detected loop had a measured length of 946 bp, in excellent agreement with the separation between the binding sites at positions 8342 and 9287 bp (a distance of 945 bp). ( B ) Sau3AI on the LBAC-A template, which has 55 recognition sites. ( C ) Control experiments: (i) the two-site enzyme Sau3AI was tested on bacteriophage phiX174 DNA, which contains no copies of its recognition sequence; (ii) the one-site enzyme HaeIII was tested on LBAC-B DNA containing 36 copies of its recognition sequence; (iii) the two-site enzyme SfiI was tested on LBAC-B DNA containing one copy of its recognition sequence and (iv) LBAC-B DNA, containing 26 FokI binding sites, incubated in the FokI reaction buffer, but no FokI added.

    Article Snippet: Endonucleases BamHI, BpmI, BsgI, BspMI, BstNI, EcoRI, EcoRV, FokI, HaeIII, HpaII, MboII, MspI, NarI, NaeI, SacII, Sau3AI, SfiI and SgrAI were obtained from New England Biolabs (NEB).

    Techniques: Binding Assay, Sequencing, Incubation

    Restriction endonuclease map and construction of plasmids used to make allelic exchange mutants and to complement LOS genes in H. somnus ). pCAAT was digested with Eco RI to obtain the 3.9-kb fragment, and pGEM-3Z was digested with Hin cII to obtain a 2.7-kb fragment. These fragments were ligated together to obtain 6.6-kb plasmid pCAAT-SalI, which was further digested with Sal I- Bsg I to obtain a 6.1-kb fragment with a 630-bp deletion in lob-2A . The 1.2-kb Sma I fragment from pUC4-kixx containing the Tn 5 Km r cassette was then ligated into the Sal I- Bsg I site of pCAAT-SalI to obtain 7.3-kb plasmid pCAATΔlob2A. The 8.8-kb plasmid pLSGA was constructed by digesting pCAAT with Eco RI and cloning the 3.9-kb fragment into the Sma I site of pLS88. pLSGA was then cut with Xba I and Hpa I to remove part of lob-1 and the non-LOS afu -like genes, and the 6.84-kb plasmid was ligated to itself. All ligations were done following blunt ending of the plasmids.

    Journal: Infection and Immunity

    Article Title: Molecular Cloning and Mutagenesis of a DNA Locus Involved in Lipooligosaccharide Biosynthesis in Haemophilus somnus

    doi:

    Figure Lengend Snippet: Restriction endonuclease map and construction of plasmids used to make allelic exchange mutants and to complement LOS genes in H. somnus ). pCAAT was digested with Eco RI to obtain the 3.9-kb fragment, and pGEM-3Z was digested with Hin cII to obtain a 2.7-kb fragment. These fragments were ligated together to obtain 6.6-kb plasmid pCAAT-SalI, which was further digested with Sal I- Bsg I to obtain a 6.1-kb fragment with a 630-bp deletion in lob-2A . The 1.2-kb Sma I fragment from pUC4-kixx containing the Tn 5 Km r cassette was then ligated into the Sal I- Bsg I site of pCAAT-SalI to obtain 7.3-kb plasmid pCAATΔlob2A. The 8.8-kb plasmid pLSGA was constructed by digesting pCAAT with Eco RI and cloning the 3.9-kb fragment into the Sma I site of pLS88. pLSGA was then cut with Xba I and Hpa I to remove part of lob-1 and the non-LOS afu -like genes, and the 6.84-kb plasmid was ligated to itself. All ligations were done following blunt ending of the plasmids.

    Article Snippet: Bsg I and Hha I methylase were purchased from New England Biolabs (Beverly, Mass.).

    Techniques: Plasmid Preparation, Construct, Clone Assay