ecorv  (Roche)


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    Structured Review

    Roche ecorv
    Mobility of ICE Kp1 in strain NTUH-K2044. (A) PCR analysis of the location of ICE Kp1 in strains NTUH-K2044 and NTUH-K2044 ICE Kp1 − . (B) Southern hybridization of <t>EcoRV-digested</t> <t>DNA</t> from strains NTUH-K2044 and NTUH-K2044 ICE Kp1 − with asn
    Ecorv, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Characterization of Integrative and Conjugative Element ICEKp1-Associated Genomic Heterogeneity in a Klebsiella pneumoniae Strain Isolated from a Primary Liver Abscess ▿"

    Article Title: Characterization of Integrative and Conjugative Element ICEKp1-Associated Genomic Heterogeneity in a Klebsiella pneumoniae Strain Isolated from a Primary Liver Abscess ▿

    Journal:

    doi: 10.1128/JB.01219-07

    Mobility of ICE Kp1 in strain NTUH-K2044. (A) PCR analysis of the location of ICE Kp1 in strains NTUH-K2044 and NTUH-K2044 ICE Kp1 − . (B) Southern hybridization of EcoRV-digested DNA from strains NTUH-K2044 and NTUH-K2044 ICE Kp1 − with asn
    Figure Legend Snippet: Mobility of ICE Kp1 in strain NTUH-K2044. (A) PCR analysis of the location of ICE Kp1 in strains NTUH-K2044 and NTUH-K2044 ICE Kp1 − . (B) Southern hybridization of EcoRV-digested DNA from strains NTUH-K2044 and NTUH-K2044 ICE Kp1 − with asn

    Techniques Used: Polymerase Chain Reaction, Hybridization

    2) Product Images from "DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway"

    Article Title: DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki565

    Measured cleavage rate versus DNA tension. ( a ) The effect of tension on cleavage of linearized pCco5 by EcoRV (number of recognition sites n = 1). EcoRV concentration was 25 nM in terms of dimers. The total amount of cutting events was 68. ( b ) EcoRV on Lambda phage DNA ( n = 21), 32 cutting events. ( c ) Tension dependence on DNA cleavage by BamHI (300 nM) on the pCco5 derivative containing a single recognition site (squares, 78 events) and on Lambda phage DNA with five BamHI sites (triangles, 28 events) using 2.5 nM BamHI. Each point consists of at least 6 cleavage events. Vertical error bars represent the standard error of the mean rate. Horizontal error bars are the standard deviation of the combined DNA tensions (a result of the binning). The data in (a and b) are fitted to the described model (normalized χ 2 are 0.6 and 0.2, respectively). The BamHI rates in (c) do not significantly vary with DNA tension and were fitted with constant values [normalized χ 2 are 0.8 (hydrolysis, green line) and 1.6 (diffusion, blue line)].
    Figure Legend Snippet: Measured cleavage rate versus DNA tension. ( a ) The effect of tension on cleavage of linearized pCco5 by EcoRV (number of recognition sites n = 1). EcoRV concentration was 25 nM in terms of dimers. The total amount of cutting events was 68. ( b ) EcoRV on Lambda phage DNA ( n = 21), 32 cutting events. ( c ) Tension dependence on DNA cleavage by BamHI (300 nM) on the pCco5 derivative containing a single recognition site (squares, 78 events) and on Lambda phage DNA with five BamHI sites (triangles, 28 events) using 2.5 nM BamHI. Each point consists of at least 6 cleavage events. Vertical error bars represent the standard error of the mean rate. Horizontal error bars are the standard deviation of the combined DNA tensions (a result of the binning). The data in (a and b) are fitted to the described model (normalized χ 2 are 0.6 and 0.2, respectively). The BamHI rates in (c) do not significantly vary with DNA tension and were fitted with constant values [normalized χ 2 are 0.8 (hydrolysis, green line) and 1.6 (diffusion, blue line)].

    Techniques Used: Concentration Assay, Standard Deviation, Diffusion-based Assay

    Crystal structures of specific enzyme–DNA complexes of BamHI (1) ( 23 ) and EcoRV (2) ( 2 , 18 ). The DNA configuration within both complexes is shown separately, (3) and (4), respectively. While BamHI does not distort its recognition site, EcoRV induces a 50° kink located at the center base pair step.
    Figure Legend Snippet: Crystal structures of specific enzyme–DNA complexes of BamHI (1) ( 23 ) and EcoRV (2) ( 2 , 18 ). The DNA configuration within both complexes is shown separately, (3) and (4), respectively. While BamHI does not distort its recognition site, EcoRV induces a 50° kink located at the center base pair step.

    Techniques Used:

    3) Product Images from "BDNF-Live-Exon-Visualization (BLEV) Allows Differential Detection of BDNF Transcripts in vitro and in vivo"

    Article Title: BDNF-Live-Exon-Visualization (BLEV) Allows Differential Detection of BDNF Transcripts in vitro and in vivo

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00325

    Generation of the BLEV reporter mouse. (A) Schematic drawing of the Bdnf gene (Aid et al., 2007 ) and the BDNF vector construct used for homologous recombination. Crossing-over events are indicated by red dotted lines and crosses. The 5' and 3' parts for homologous recombination are marked in red. (B) Bdnf gene construct depicting insertion sites of the BLEV construct. Arrows indicate the EcoRV restriction sites (RS) used for Southern blot. Black arrows indicate EcoRV RS cutting the WT and the transgenic sequence, red arrows cut only the transgenic sequence. (C) Southern blot of a positively transfected ES cell clone. The positions of the four Southern blot probes (colored lines: green, blue, orange, and pink) are shown under the schematic drawing of the BDNF construct. Probe 1, specific for the 5′-end: WT band 9.8 kb, transgenic allele 3 kb; probe 2, covering parts of exon-IV transgene: WT band 9.8 kb, transgenic allele 5.3 kb; probe 3, covering parts of the exon-VI transgene: WT band 9.8 kb, transgenic allele 6.3 kb; probe 4, specific for the 3′-end: WT and transgenic allele 12.7 kb. Note that the size of the drawn construct and probes does not reflect the real size of the DNA fragments obtained by Southern blot. For original Blots see Supplementary Figure 1 .
    Figure Legend Snippet: Generation of the BLEV reporter mouse. (A) Schematic drawing of the Bdnf gene (Aid et al., 2007 ) and the BDNF vector construct used for homologous recombination. Crossing-over events are indicated by red dotted lines and crosses. The 5' and 3' parts for homologous recombination are marked in red. (B) Bdnf gene construct depicting insertion sites of the BLEV construct. Arrows indicate the EcoRV restriction sites (RS) used for Southern blot. Black arrows indicate EcoRV RS cutting the WT and the transgenic sequence, red arrows cut only the transgenic sequence. (C) Southern blot of a positively transfected ES cell clone. The positions of the four Southern blot probes (colored lines: green, blue, orange, and pink) are shown under the schematic drawing of the BDNF construct. Probe 1, specific for the 5′-end: WT band 9.8 kb, transgenic allele 3 kb; probe 2, covering parts of exon-IV transgene: WT band 9.8 kb, transgenic allele 5.3 kb; probe 3, covering parts of the exon-VI transgene: WT band 9.8 kb, transgenic allele 6.3 kb; probe 4, specific for the 3′-end: WT and transgenic allele 12.7 kb. Note that the size of the drawn construct and probes does not reflect the real size of the DNA fragments obtained by Southern blot. For original Blots see Supplementary Figure 1 .

    Techniques Used: Plasmid Preparation, Construct, Homologous Recombination, Southern Blot, Transgenic Assay, Sequencing, Transfection

    Related Articles

    Isolation:

    Article Title: Insertional Mutagenesis and Deep Profiling Reveals Gene Hierarchies and a Myc/p53-Dependent Bottleneck in Lymphomagenesis
    Article Snippet: DNA extraction DNA was extracted from approximately 20 mg of frozen enlarged lymphoid/tumour tissue using Gentra Puregene Genomic DNA Purification Kit (Qiagen, UK) according to the manufacturer's instructions. .. Isolation of retroviral insertion sites Isolation of the retroviral insertion sites from the tissues was performed using splinkerette PCR to produce barcoded PCR products that were pooled and sequenced on 454 GS-FLX sequencers (Roche Diagnostics platform) as described previously , . ..

    Article Title: PAXX and XLF DNA repair factors are functionally redundant in joining DNA breaks in a G1-arrested progenitor B-cell line
    Article Snippet: Genomic DNA was isolated from individual clones as described above for v-Abl cells. .. RNA was isolated from G1-arrested Abl pro–B-cell RNA (Tripure Reagent; Roche). .. PAXX cDNA was amplified ( ) from the resulting cDNA pool (Invitrogen Superscript Plus II) and ligated into EcoRV-digested and CIP-treated pBluescript II (KS+).

    Polymerase Chain Reaction:

    Article Title: Insertional Mutagenesis and Deep Profiling Reveals Gene Hierarchies and a Myc/p53-Dependent Bottleneck in Lymphomagenesis
    Article Snippet: DNA extraction DNA was extracted from approximately 20 mg of frozen enlarged lymphoid/tumour tissue using Gentra Puregene Genomic DNA Purification Kit (Qiagen, UK) according to the manufacturer's instructions. .. Isolation of retroviral insertion sites Isolation of the retroviral insertion sites from the tissues was performed using splinkerette PCR to produce barcoded PCR products that were pooled and sequenced on 454 GS-FLX sequencers (Roche Diagnostics platform) as described previously , . ..

    Amplification:

    Article Title: Assembly of an active translation initiation factor complex by a viral protein
    Article Snippet: To add a poly-histidine tag to the ICP6 N terminus, ICP6 coding sequences contained in the NotI–EcoRV segment of the genomic EcoRI A fragment were isolated and subcloned into pBSΔXho that had been previously digested with XbaI, end-filled with Klenow, then digested with NotI. .. The ICP6 gene was amplified from this plasmid using the Taq expand amplification kit (Roche) along with a primer adding sequences encoding a His tag at the 5′ end (5′-ATCTCGAGAATTCCATGGCCAGCC GCCCAGCCGCATCC-3′) and a primer introducing a SpeI site at the 3′ end (5′-GTACCGACTAGTTCACAGCGCGCAGCT CATGCAGAC-3′). .. Spe1/Not1 digestion was used to clone the amplified sequence into the commercial vector pFASTBAC (Invitrogen). pFASTBAC-His-ICP6 was then digested with PvuII and religated to generate pFASTBAC-His-ICP6-PvuII, which lacks 341 nucleotides in the middle region of the PCR-amplified ICP6.

    Plasmid Preparation:

    Article Title: Assembly of an active translation initiation factor complex by a viral protein
    Article Snippet: To add a poly-histidine tag to the ICP6 N terminus, ICP6 coding sequences contained in the NotI–EcoRV segment of the genomic EcoRI A fragment were isolated and subcloned into pBSΔXho that had been previously digested with XbaI, end-filled with Klenow, then digested with NotI. .. The ICP6 gene was amplified from this plasmid using the Taq expand amplification kit (Roche) along with a primer adding sequences encoding a His tag at the 5′ end (5′-ATCTCGAGAATTCCATGGCCAGCC GCCCAGCCGCATCC-3′) and a primer introducing a SpeI site at the 3′ end (5′-GTACCGACTAGTTCACAGCGCGCAGCT CATGCAGAC-3′). .. Spe1/Not1 digestion was used to clone the amplified sequence into the commercial vector pFASTBAC (Invitrogen). pFASTBAC-His-ICP6 was then digested with PvuII and religated to generate pFASTBAC-His-ICP6-PvuII, which lacks 341 nucleotides in the middle region of the PCR-amplified ICP6.

    Article Title: Localization of the Brainstem GABAergic Neurons Controlling Paradoxical (REM) Sleep
    Article Snippet: .. Fos immunohistochemistry combined with GAD67 mRNA in situ hybridization As described before , the recombinant plasmid containing the GAD67 cDNA was linearised using EcoRV and transcribed using SP6 RNA polymerase and a nonradioactive RNA labeling kit (Roche Diagnostic, Mannheim, Germany) for the antisense probe. .. The brain sections were successively incubated in a rabbit antiserum to Fos (1∶4000; Ab-5; Oncogene, CA, USA) in 10 mM PB containing 0.9% NaCl and 0.3% Triton-100× (PBST) for 18 h at room temperature; a biotinylated goat antirabbit IgG solution (1∶1000; Vector Laboratories, Burlingame, CA, USA); and an ABC-HRP solution (1∶1000; Elite kit, Vector Laboratories) both for 90 min at room temperature.

    Immunohistochemistry:

    Article Title: Localization of the Brainstem GABAergic Neurons Controlling Paradoxical (REM) Sleep
    Article Snippet: .. Fos immunohistochemistry combined with GAD67 mRNA in situ hybridization As described before , the recombinant plasmid containing the GAD67 cDNA was linearised using EcoRV and transcribed using SP6 RNA polymerase and a nonradioactive RNA labeling kit (Roche Diagnostic, Mannheim, Germany) for the antisense probe. .. The brain sections were successively incubated in a rabbit antiserum to Fos (1∶4000; Ab-5; Oncogene, CA, USA) in 10 mM PB containing 0.9% NaCl and 0.3% Triton-100× (PBST) for 18 h at room temperature; a biotinylated goat antirabbit IgG solution (1∶1000; Vector Laboratories, Burlingame, CA, USA); and an ABC-HRP solution (1∶1000; Elite kit, Vector Laboratories) both for 90 min at room temperature.

    In Situ Hybridization:

    Article Title: Localization of the Brainstem GABAergic Neurons Controlling Paradoxical (REM) Sleep
    Article Snippet: .. Fos immunohistochemistry combined with GAD67 mRNA in situ hybridization As described before , the recombinant plasmid containing the GAD67 cDNA was linearised using EcoRV and transcribed using SP6 RNA polymerase and a nonradioactive RNA labeling kit (Roche Diagnostic, Mannheim, Germany) for the antisense probe. .. The brain sections were successively incubated in a rabbit antiserum to Fos (1∶4000; Ab-5; Oncogene, CA, USA) in 10 mM PB containing 0.9% NaCl and 0.3% Triton-100× (PBST) for 18 h at room temperature; a biotinylated goat antirabbit IgG solution (1∶1000; Vector Laboratories, Burlingame, CA, USA); and an ABC-HRP solution (1∶1000; Elite kit, Vector Laboratories) both for 90 min at room temperature.

    Recombinant:

    Article Title: Localization of the Brainstem GABAergic Neurons Controlling Paradoxical (REM) Sleep
    Article Snippet: .. Fos immunohistochemistry combined with GAD67 mRNA in situ hybridization As described before , the recombinant plasmid containing the GAD67 cDNA was linearised using EcoRV and transcribed using SP6 RNA polymerase and a nonradioactive RNA labeling kit (Roche Diagnostic, Mannheim, Germany) for the antisense probe. .. The brain sections were successively incubated in a rabbit antiserum to Fos (1∶4000; Ab-5; Oncogene, CA, USA) in 10 mM PB containing 0.9% NaCl and 0.3% Triton-100× (PBST) for 18 h at room temperature; a biotinylated goat antirabbit IgG solution (1∶1000; Vector Laboratories, Burlingame, CA, USA); and an ABC-HRP solution (1∶1000; Elite kit, Vector Laboratories) both for 90 min at room temperature.

    Labeling:

    Article Title: Localization of the Brainstem GABAergic Neurons Controlling Paradoxical (REM) Sleep
    Article Snippet: .. Fos immunohistochemistry combined with GAD67 mRNA in situ hybridization As described before , the recombinant plasmid containing the GAD67 cDNA was linearised using EcoRV and transcribed using SP6 RNA polymerase and a nonradioactive RNA labeling kit (Roche Diagnostic, Mannheim, Germany) for the antisense probe. .. The brain sections were successively incubated in a rabbit antiserum to Fos (1∶4000; Ab-5; Oncogene, CA, USA) in 10 mM PB containing 0.9% NaCl and 0.3% Triton-100× (PBST) for 18 h at room temperature; a biotinylated goat antirabbit IgG solution (1∶1000; Vector Laboratories, Burlingame, CA, USA); and an ABC-HRP solution (1∶1000; Elite kit, Vector Laboratories) both for 90 min at room temperature.

    Article Title: Gene Cloning, Protein Characterization, and Alteration of Product Selectivity for the Trehalulose Hydrolase and Trehalulose Synthase from "Pseudomonas mesoacidophila" MX-45 ▿" MX-45 ▿ †
    Article Snippet: The obtained 500-bp PCR fragment was inserted into the positive selection vector pJOE4786 , which was cut with EcoRV to create plasmid pHWG705. .. The inserts from pHWG705.1 and pHWG705.6 were cut out with BamHI, digoxigenin (DIG) labeled by random-primed DNA labeling (DIG High Prime; Roche Applied Science), and used as probes mutA and mutB, respectively. ..

    Mouse Assay:

    Article Title: Expression of the Mouse Pre-T Cell Receptor ? Gene Is Controlled by an Upstream Region Containing a Transcriptional Enhancer
    Article Snippet: A 0.25-kb AspI-PstI probe from the 3′ end of the 9-kb pTα fragment hybridized with 5- and 1.6-kb EcoRV fragments from the endogenous pTα gene and the transgene, respectively, and was used for transgene copy number determination by PhosphorImager (Molecular Dynamics) quantitation. .. Total RNA was extracted from various organs of 4-wk-old F1-transgenic mice, and Poly(A) RNA was prepared from 250 μg total RNA using biotin-oligo(dT)/streptavidin magnetic beads (Roche Molecular Biochemicals). .. Transgene expression was analyzed by Northern hybridization with a 5′ 1.3-kb fragment of LacZ gene.

    Magnetic Beads:

    Article Title: Expression of the Mouse Pre-T Cell Receptor ? Gene Is Controlled by an Upstream Region Containing a Transcriptional Enhancer
    Article Snippet: A 0.25-kb AspI-PstI probe from the 3′ end of the 9-kb pTα fragment hybridized with 5- and 1.6-kb EcoRV fragments from the endogenous pTα gene and the transgene, respectively, and was used for transgene copy number determination by PhosphorImager (Molecular Dynamics) quantitation. .. Total RNA was extracted from various organs of 4-wk-old F1-transgenic mice, and Poly(A) RNA was prepared from 250 μg total RNA using biotin-oligo(dT)/streptavidin magnetic beads (Roche Molecular Biochemicals). .. Transgene expression was analyzed by Northern hybridization with a 5′ 1.3-kb fragment of LacZ gene.

    Random Primed:

    Article Title: Gene Cloning, Protein Characterization, and Alteration of Product Selectivity for the Trehalulose Hydrolase and Trehalulose Synthase from "Pseudomonas mesoacidophila" MX-45 ▿" MX-45 ▿ †
    Article Snippet: The obtained 500-bp PCR fragment was inserted into the positive selection vector pJOE4786 , which was cut with EcoRV to create plasmid pHWG705. .. The inserts from pHWG705.1 and pHWG705.6 were cut out with BamHI, digoxigenin (DIG) labeled by random-primed DNA labeling (DIG High Prime; Roche Applied Science), and used as probes mutA and mutB, respectively. ..

    DNA Labeling:

    Article Title: Gene Cloning, Protein Characterization, and Alteration of Product Selectivity for the Trehalulose Hydrolase and Trehalulose Synthase from "Pseudomonas mesoacidophila" MX-45 ▿" MX-45 ▿ †
    Article Snippet: The obtained 500-bp PCR fragment was inserted into the positive selection vector pJOE4786 , which was cut with EcoRV to create plasmid pHWG705. .. The inserts from pHWG705.1 and pHWG705.6 were cut out with BamHI, digoxigenin (DIG) labeled by random-primed DNA labeling (DIG High Prime; Roche Applied Science), and used as probes mutA and mutB, respectively. ..

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    Roche ecorv
    Measured cleavage rate versus DNA tension. ( a ) The effect of tension on cleavage of linearized pCco5 by <t>EcoRV</t> (number of recognition sites n = 1). EcoRV concentration was 25 nM in terms of dimers. The total amount of cutting events was 68. ( b ) EcoRV on Lambda phage DNA ( n = 21), 32 cutting events. ( c ) Tension dependence on DNA cleavage by <t>BamHI</t> (300 nM) on the pCco5 derivative containing a single recognition site (squares, 78 events) and on Lambda phage DNA with five BamHI sites (triangles, 28 events) using 2.5 nM BamHI. Each point consists of at least 6 cleavage events. Vertical error bars represent the standard error of the mean rate. Horizontal error bars are the standard deviation of the combined DNA tensions (a result of the binning). The data in (a and b) are fitted to the described model (normalized χ 2 are 0.6 and 0.2, respectively). The BamHI rates in (c) do not significantly vary with DNA tension and were fitted with constant values [normalized χ 2 are 0.8 (hydrolysis, green line) and 1.6 (diffusion, blue line)].
    Ecorv, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ecorv/product/Roche
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ecorv - by Bioz Stars, 2021-06
    86/100 stars
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    Measured cleavage rate versus DNA tension. ( a ) The effect of tension on cleavage of linearized pCco5 by EcoRV (number of recognition sites n = 1). EcoRV concentration was 25 nM in terms of dimers. The total amount of cutting events was 68. ( b ) EcoRV on Lambda phage DNA ( n = 21), 32 cutting events. ( c ) Tension dependence on DNA cleavage by BamHI (300 nM) on the pCco5 derivative containing a single recognition site (squares, 78 events) and on Lambda phage DNA with five BamHI sites (triangles, 28 events) using 2.5 nM BamHI. Each point consists of at least 6 cleavage events. Vertical error bars represent the standard error of the mean rate. Horizontal error bars are the standard deviation of the combined DNA tensions (a result of the binning). The data in (a and b) are fitted to the described model (normalized χ 2 are 0.6 and 0.2, respectively). The BamHI rates in (c) do not significantly vary with DNA tension and were fitted with constant values [normalized χ 2 are 0.8 (hydrolysis, green line) and 1.6 (diffusion, blue line)].

    Journal: Nucleic Acids Research

    Article Title: DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway

    doi: 10.1093/nar/gki565

    Figure Lengend Snippet: Measured cleavage rate versus DNA tension. ( a ) The effect of tension on cleavage of linearized pCco5 by EcoRV (number of recognition sites n = 1). EcoRV concentration was 25 nM in terms of dimers. The total amount of cutting events was 68. ( b ) EcoRV on Lambda phage DNA ( n = 21), 32 cutting events. ( c ) Tension dependence on DNA cleavage by BamHI (300 nM) on the pCco5 derivative containing a single recognition site (squares, 78 events) and on Lambda phage DNA with five BamHI sites (triangles, 28 events) using 2.5 nM BamHI. Each point consists of at least 6 cleavage events. Vertical error bars represent the standard error of the mean rate. Horizontal error bars are the standard deviation of the combined DNA tensions (a result of the binning). The data in (a and b) are fitted to the described model (normalized χ 2 are 0.6 and 0.2, respectively). The BamHI rates in (c) do not significantly vary with DNA tension and were fitted with constant values [normalized χ 2 are 0.8 (hydrolysis, green line) and 1.6 (diffusion, blue line)].

    Article Snippet: Plasmid pCco5 DNA (6538 bp; gift from W. Reijnders, Vrije Universiteit, Amsterdam) was utilized for the single cleavage site experiments on EcoRV and BamHI, whereas for the multiple site experiments Lambda phage DNA (48 502 bp; Roche GmbH, Germany) was used.

    Techniques: Concentration Assay, Standard Deviation, Diffusion-based Assay

    Crystal structures of specific enzyme–DNA complexes of BamHI (1) ( 23 ) and EcoRV (2) ( 2 , 18 ). The DNA configuration within both complexes is shown separately, (3) and (4), respectively. While BamHI does not distort its recognition site, EcoRV induces a 50° kink located at the center base pair step.

    Journal: Nucleic Acids Research

    Article Title: DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway

    doi: 10.1093/nar/gki565

    Figure Lengend Snippet: Crystal structures of specific enzyme–DNA complexes of BamHI (1) ( 23 ) and EcoRV (2) ( 2 , 18 ). The DNA configuration within both complexes is shown separately, (3) and (4), respectively. While BamHI does not distort its recognition site, EcoRV induces a 50° kink located at the center base pair step.

    Article Snippet: Plasmid pCco5 DNA (6538 bp; gift from W. Reijnders, Vrije Universiteit, Amsterdam) was utilized for the single cleavage site experiments on EcoRV and BamHI, whereas for the multiple site experiments Lambda phage DNA (48 502 bp; Roche GmbH, Germany) was used.

    Techniques:

    Generation of the BLEV reporter mouse. (A) Schematic drawing of the Bdnf gene (Aid et al., 2007 ) and the BDNF vector construct used for homologous recombination. Crossing-over events are indicated by red dotted lines and crosses. The 5' and 3' parts for homologous recombination are marked in red. (B) Bdnf gene construct depicting insertion sites of the BLEV construct. Arrows indicate the EcoRV restriction sites (RS) used for Southern blot. Black arrows indicate EcoRV RS cutting the WT and the transgenic sequence, red arrows cut only the transgenic sequence. (C) Southern blot of a positively transfected ES cell clone. The positions of the four Southern blot probes (colored lines: green, blue, orange, and pink) are shown under the schematic drawing of the BDNF construct. Probe 1, specific for the 5′-end: WT band 9.8 kb, transgenic allele 3 kb; probe 2, covering parts of exon-IV transgene: WT band 9.8 kb, transgenic allele 5.3 kb; probe 3, covering parts of the exon-VI transgene: WT band 9.8 kb, transgenic allele 6.3 kb; probe 4, specific for the 3′-end: WT and transgenic allele 12.7 kb. Note that the size of the drawn construct and probes does not reflect the real size of the DNA fragments obtained by Southern blot. For original Blots see Supplementary Figure 1 .

    Journal: Frontiers in Molecular Neuroscience

    Article Title: BDNF-Live-Exon-Visualization (BLEV) Allows Differential Detection of BDNF Transcripts in vitro and in vivo

    doi: 10.3389/fnmol.2018.00325

    Figure Lengend Snippet: Generation of the BLEV reporter mouse. (A) Schematic drawing of the Bdnf gene (Aid et al., 2007 ) and the BDNF vector construct used for homologous recombination. Crossing-over events are indicated by red dotted lines and crosses. The 5' and 3' parts for homologous recombination are marked in red. (B) Bdnf gene construct depicting insertion sites of the BLEV construct. Arrows indicate the EcoRV restriction sites (RS) used for Southern blot. Black arrows indicate EcoRV RS cutting the WT and the transgenic sequence, red arrows cut only the transgenic sequence. (C) Southern blot of a positively transfected ES cell clone. The positions of the four Southern blot probes (colored lines: green, blue, orange, and pink) are shown under the schematic drawing of the BDNF construct. Probe 1, specific for the 5′-end: WT band 9.8 kb, transgenic allele 3 kb; probe 2, covering parts of exon-IV transgene: WT band 9.8 kb, transgenic allele 5.3 kb; probe 3, covering parts of the exon-VI transgene: WT band 9.8 kb, transgenic allele 6.3 kb; probe 4, specific for the 3′-end: WT and transgenic allele 12.7 kb. Note that the size of the drawn construct and probes does not reflect the real size of the DNA fragments obtained by Southern blot. For original Blots see Supplementary Figure 1 .

    Article Snippet: In brief, isolated DNA was digested using EcoRV and SalI (Roche).

    Techniques: Plasmid Preparation, Construct, Homologous Recombination, Southern Blot, Transgenic Assay, Sequencing, Transfection