bsgi  (New England Biolabs)

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

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

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

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