eagi  (New England Biolabs)

Journal: Biotechnology and bioengineering

Article Title: Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA

doi: 10.1002/bit.23224

Figure Lengend Snippet: S1 nuclease converts supercoiled plasmid DNA to nicked circular and linear forms through digestion at a variety of locations in the plasmid. (A) Two μg of supercoiled pRH04.152-rbsB was incubated at 22°C (top) or 37°C (bottom) with different amounts of S1 nuclease for different lengths of time and then analyzed by agarose gel electrophoresis. The sixth and seventh lanes contain undigested supercoiled control and SpeI-digested linear control, respectively. The band that runs slower than the linear band was presumed to correspond to nicked circular plasmid. In the thirteenth lane, after the DNA was digested with 10 units for 24 hours an additional 10 units of S1 nuclease was added and the DNA was incubated for an additional hour. The first and last lanes contain the λDNA/HindIII molecular weight standards. (B) Plasmids with or without the f1 origin (pDIMC8-pfMBP and pRH04-pfMBP, respectively) were incubated with or without S1 nuclease, followed by incubation with or without NcoI, EagI, and SacII and then analyzed by agarose gel electrophoresis. (C) Plasmids with or without the f1 origin (pDIMC8-rbsb and pRH04-rbsb, respectively) were incubated with S1 nuclease or DNaseI, followed by incubation with XmnI and analyzed by agarose gel electrophoresis.

Article Snippet: This linear DNA was digested with restriction enzymes NcoI, EagI, SacI or XmnI under the recommended conditions (New England Biolabs) for 1 hour at 37°C and analyzed by agarose gel electrophoresis on a 0.8% agarose, TAE gel.

Techniques: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Molecular Weight

Journal: Journal of Biological Engineering

Article Title: End-to-end automated microfluidic platform for synthetic biology: from design to functional analysis

doi: 10.1186/s13036-016-0024-5

Figure Lengend Snippet: Construction in yeast of a promoter library. a Promoter library schematic. b Gel electrophoresis image of amplified promoters: lane 1, Gal-250; lane 2, Gal-100; lane 3, Leu-250; lane 4, spo-250; lane 5, spo-100; lane 6, tef-250; lane 7, tef-100. c Gel electrophoresis image of: lane 1, pRS426; lane 2, pRS426-yeGFP EagI digest. M is GeneRuler 1 kb Plus DNA Ladder (Thermo Scientific). d Schematic of reagent transfers through the microfluidic chip. The green input wells contain a mixture of different promoters and digested plasmid pRS426 with Salomon sperm DNA as a carrier. The yellow input wells contain S. cerevisiae competent cells. Arrows show pathways of reagent transfer on-chip according to the automated protocol

Article Snippet: Digestion of pRS426-yeGFP plasmid by EagI 50 μL EagI (NEB) digestion reactions consisting of 20 μL purified plasmid pRS426 (100 ng/μL), 5 μL CutSmart™ Buffer, EagI-HF (20 units/μL) 1 μL and 24 μL deionized water.

Techniques: Nucleic Acid Electrophoresis, Amplification, Chromatin Immunoprecipitation, Plasmid Preparation

Journal: Frontiers in Microbiology

Article Title: Novel Multidrug-Resistant Enterococcal Mobile Linear Plasmid pELF1 Encoding vanA and vanM Gene Clusters From a Japanese Vancomycin-Resistant Enterococci Isolate

doi: 10.3389/fmicb.2019.02568

Figure Lengend Snippet: Genetic structure of vancomycin resistance, other antimicrobial drug resistance genes, and pELF1. (A) The genetic structure of vancomycin resistance genes and other antimicrobial drug resistance genes is shown. Elements IS 1216 V and IS 1542 were inserted in the Tn 1546 -like element harboring vanA gene cluster. In contrast, two IS 1216E elements were located in the same direction at both ends of the vanM gene cluster. aadE , sat4 , aphA-3 , and ermB were located in pELF1 in this order. sat4 was interrupted by IS Efm1 and IS Efa11 . (B) The schematic structure of pLFE1 is shown. The left terminal end formed a hairpin structure. On the other hand, the right end is presumed to be of the invertron type. Drug resistance genes as well as presumed replication machinery and transfer machinery genes are shown. The schematic structure was prepared using easyfig ( Sullivan et al., 2011 ). (C) Restriction map of pELF1 (143.3 kb) is shown. A single Sac I, Sal I, and Sma I site each and two Eag I sites are present in pELF1. The fragments produced by these for restriction enzymes are denoted by letters.

Article Snippet: To determine the physical map of pELF1, the excised plugs containing pELF1 DNAs were separately digested at 37°C for 24 h with 20 U of Sal I-HF (New England BioLabs, Ipswich, MA, United States), at 25°C for 24 h with 100 U of Sma I (New England BioLabs), at 37°C for 24 h with 150 U of Eag I-HF (New England BioLabs), and at 37°C for 24 h with 150 U of Sac I-HF (New England BioLabs).

Techniques: Produced

Journal: Frontiers in Microbiology

Article Title: Novel Multidrug-Resistant Enterococcal Mobile Linear Plasmid pELF1 Encoding vanA and vanM Gene Clusters From a Japanese Vancomycin-Resistant Enterococci Isolate

doi: 10.3389/fmicb.2019.02568

Figure Lengend Snippet: PFGE of the restriction fragments of pELF1 with proteinase K treatment. Lanes: Low Range PFG Marker; proteinase K-treatment only; proteinase K-treated Sal I digests; proteinase K-treated Sma I digests; proteinase K-treated Eag I digests. N. D., not digested.

Article Snippet: To determine the physical map of pELF1, the excised plugs containing pELF1 DNAs were separately digested at 37°C for 24 h with 20 U of Sal I-HF (New England BioLabs, Ipswich, MA, United States), at 25°C for 24 h with 100 U of Sma I (New England BioLabs), at 37°C for 24 h with 150 U of Eag I-HF (New England BioLabs), and at 37°C for 24 h with 150 U of Sac I-HF (New England BioLabs).

Techniques: Marker

Journal: Aging Cell

Article Title: Analysis of somatic mutations identifies signs of selection during in vitro aging of primary dermal fibroblasts, et al. Analysis of somatic mutations identifies signs of selection during in vitro aging of primary dermal fibroblasts

doi: 10.1111/acel.13010

Figure Lengend Snippet: CDKN2A gene and its expression in the presence or absence of the c.53C > T mutation. (a) Schematic representation of the genomic structure and transcripts of the CDKN2A gene locus with c.53C > T mutation and primer locations. The exons are shown as rectangles and primers as arrows. Primers 1 and 5 were used for RFLP analysis (see figure c), primers 3 and 4 were used for ddPCR analysis (see figure b), and primers 2 and 5 were used for additional ddPCR analysis (see Figure S1 ). An EagI restriction site is present in a wild‐type carrier, but absent in the individual with the c.53C > T mutation. The size of the locus, transcripts, exons, and introns is not shown to scale. Of note, the size of the intron between exon 1a and exon 2 is approximately 3.5 kb. (b) Comparison of CDKN2A transcript copies after normalization to GAPDH in early and late passage primary fibroblasts from young and advanced age healthy subjects without the c.53C > T mutation and in early and late passage primary fibroblasts from the XPA patient where the c.53C > T mutation is present at an allele frequency of 55.3% in the late passage. Primers 3 and 4 were used for this ddPCR analysis. (c) RFLP analysis of CDKN2A transcript in early and late passage primary fibroblasts from young age healthy and advanced age healthy subjects without the c.53C > T mutation and in early and late passage primary fibroblasts from XPA patient where the c.53C > T mutation is present at an allele frequency of 55.3% in the late passage only. Primers 1 and 5 were first used to amplify the cDNA followed by digestion with restriction enzyme EagI (indicated by +). Part of the amplified sample was used as an undigested control (indicated by −). Undigested PCR product is 658 bp in size, while digested fragments are 274 bp and 384 bp in size

Article Snippet: After clean‐up with QIAquick PCR purification kit (Qiagen) according to the manufacturer's recommendations, 25% of the PCR product was digested using EagI restriction enzyme (10 U/reaction; New England Biolabs) and visualized on 2% agarose gel.

Techniques: Expressing, Mutagenesis, Amplification, Polymerase Chain Reaction