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  • 99
    New England Biolabs restriction endonucleases ecorv
    Controlled release of USPIO micelles by environmental triggers . (A) Self-assembly of <t>EcoRV-sensitive</t> ssDNA-USPIO clusters, and subsequent enzymatic treatment results in measurable changes in R 2 relaxation coefficient relative to initial values. Following EcoRV treatment, R 2 values return to baseline, a phenomenon that is in significant contrast to the effects of <t>EcoRI</t> treatment of the same clusters (n = 6). * p
    Restriction Endonucleases Ecorv, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 325 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher ecorv
    Restriction enzyme-digested fragments of the genomic <t>DNA</t> of A. hydrophila -phage 2. Footnote: Lane M: 1kb Plus Opti-DNA Marker (ABM, Canada); Lane L1: genomic DNA of Φ2; Lanes L2–L8: genomic DNA of Φ2 digested with <t>EcoRV;</t> EcoRI; Ncol; SalI; MspI; XmnI; KpnI, restriction enzymes respectively.
    Ecorv, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 2031 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega ecorv
    Tryptase genotyping in healthy subjects. α/β-Tryptase genotyping was performed with <t>gDNA</t> obtained from SMCs of 24 subjects. <t>EcoRV</t> digestions of PCR amplimers labeled during the final cycle with DY682-labeled 3′-primer were performed. Gel images are shown for 3 of the 24 samples analyzed, 2 with a β:α ratio of 3∶1 and 1 with a ratio of 2∶2. The 17 samples in which the α-tryptase gene was detected are shown in the plot, where those with a 2∶2 or 3∶1 genotype are grouped together. Data is stored in S4 Table .
    Ecorv, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 546 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ecorv  (Roche)
    92
    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: 92/100, based on 161 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc 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 Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GenScript 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 GenScript, used in various techniques. Bioz Stars score: 92/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs ecorv restriction sites
    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 Restriction Sites, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher eco32i
    Restriction patterns of DNA extracted from purified virions cleaved with the selected REases. Panel A: HindIII (H), <t>Eco32I</t> (E32), SalI (S) and EcoRI (EI). ND, undigested DNA. M, GeneRuler 100- to 10,000-bp size marker. λ/H, λ DNA cleaved with HindIII. ~14 kb restriction fragment of EcoR32I digested virion DNA is marked with a triangle. Panel B: Arrows indicate restriction fragments assigned to ФAH14a (left) and ФAH14b (right) genomic DNA, obtained by SalI digestion.
    Eco32i, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore ecor v
    Restriction patterns of DNA extracted from purified virions cleaved with the selected REases. Panel A: HindIII (H), <t>Eco32I</t> (E32), SalI (S) and EcoRI (EI). ND, undigested DNA. M, GeneRuler 100- to 10,000-bp size marker. λ/H, λ DNA cleaved with HindIII. ~14 kb restriction fragment of EcoR32I digested virion DNA is marked with a triangle. Panel B: Arrows indicate restriction fragments assigned to ФAH14a (left) and ФAH14b (right) genomic DNA, obtained by SalI digestion.
    Ecor V, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs ecorv hf
    (A) AAV vector maps depicting AAV-P Tight -Cas9 and AAV-gRNA/rtTA. AAV-P Tight -Cas9 consists of a Cas9 transgene under the control of a Dox inducible Tight promoter. AAV-gRNA/rtTA consists of a gRNA expression cassette and a rtTA (Tet-On Advanced) transgene controlled by a CMV promoter. It also is designed to express GFP via an IRES element following the rtTA reading frame. (B) ICC for Cas9 and GFP was performed on 293FT cells transduced by AAV-P Tight -Cas9 and AAV-gRNA/rtTA viruses in the presence or absence of Dox. Native GFP expression is visible in virtually all of the cells (i, iii). Cas9 expression is robustly induced in the presence of Dox (ii), compared to the no Dox condition (iv). Representative images are shown. The experiment was repeated twice with similar results. (C) Diagram depicting the approximate location of where the Tet2 gRNA targets the Tet2 locus. Underlined nucleotides indicate the sequence of the Tet2 gRNA. Location of the <t>EcoRV</t> site and PAM sequence are denoted. (D) An approximate 460 bps region of the Tet2 locus that includes the site targeted for editing via the gRNA Tet2 , was <t>PCR</t> amplified from N2A genomic DNA and electrophoresed on a standard agarose gel and stained with ethidium bromide (lane 1). N2A cells were transfected with the pX330 Empty , a plasmid designed to express spCas9 and no gRNA, and 96 h later, the genomic DNA was isolated and the Tet2 locus was PCR amplified and subjected to EcoRV digestion. The PCR product was cut into two pieces of DNA as expected (lane 2). However, when N2A cells were transfected with pX330 Tet2 and similarly processed, the PCR product was incompletely digested resulting in a total of three bands on the gel - one uncut PCR product (~460 bps) and two smaller bands. In this case the genome editing was ~33%. (E) Edited DNA depicted in ( D , lane 3) was gel purified and TA cloned and 6 independent clones were sequenced. These 6 clones contained deletions which destroyed the EcoRV site.
    Ecorv Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 189 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs ecorv restriction endonuclease
    Full-length wild-type p53 binds to MYC G-quadruplex more efficiently than its isolated C-terminal and central regions The role of p53 domains in MYC G-quadruplex binding was studied by EMSA. Oligonucleotides (1 pmol) Pu52 ( A ), Pu33 ( B ) and Pu22 ( C ) were incubated with wtp53 (lanes 2–5; 50, 100, 200, 400 ng), p53-320 (lanes 6–9; 25, 50, 100, 200 ng) or p53CD (lanes 10–13; 12.5, 25, 50, 100 ng) in the presence of 10 ng of non-specific competitor <t>pBSK/EcoRV.</t> Binding of full-length wtp53, central DBD and C-terminal construct of p53 to double-stranded oligonucleotides containing p53CON was studied by EMSA. Oligonucleotides (0.25 pmol) ( D ) P1-50, ( E ) P1-30 and ( F ) P1-22 were incubated with wtp53 (lanes 2–5; 12.5, 25, 50, 100 ng), p53-320 (lanes 6–9; 6, 12.5, 25, 50 ng) or p53CD (lanes 10–13; 3, 6, 12.5, 25 ng) in the presence of 2.5 ng of non-specific competitor pBSK/EcoRV.
    Ecorv Restriction Endonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Controlled release of USPIO micelles by environmental triggers . (A) Self-assembly of EcoRV-sensitive ssDNA-USPIO clusters, and subsequent enzymatic treatment results in measurable changes in R 2 relaxation coefficient relative to initial values. Following EcoRV treatment, R 2 values return to baseline, a phenomenon that is in significant contrast to the effects of EcoRI treatment of the same clusters (n = 6). * p

    Journal: Journal of Nanobiotechnology

    Article Title: Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs)

    doi: 10.1186/1477-3155-9-7

    Figure Lengend Snippet: Controlled release of USPIO micelles by environmental triggers . (A) Self-assembly of EcoRV-sensitive ssDNA-USPIO clusters, and subsequent enzymatic treatment results in measurable changes in R 2 relaxation coefficient relative to initial values. Following EcoRV treatment, R 2 values return to baseline, a phenomenon that is in significant contrast to the effects of EcoRI treatment of the same clusters (n = 6). * p

    Article Snippet: The restriction enzymes EcoRI and EcoRV were purchased from New England Biolabs (Ipswich, MA).

    Techniques:

    Diagram of the portion of mitochondrial Cytochrome Oxidase I ( COI ) gene used to identify strain and the individual corn-strain (CS) haplotypes. The putative translational start site of the COI gene was arbitrarily designated as coordinate 0. Short block arrows indicate location and direction of the COI-893F and COI-1303R primers used for PCR amplification and DNA sequencing. Vertical lines within the COI gene identify polymorphic sites with the rice-strain (RS) specific EcoRV site identified. All polymorphic sites except 1287 were used to identify or confirm strain identity. The CS-h haplotypes were determined by the polymorphisms at site 1164 and 1287.

    Journal: Ecology and Evolution

    Article Title: Inferring the annual migration patterns of fall armyworm (Lepidoptera: Noctuidae) in the United States from mitochondrial haplotypes

    doi: 10.1002/ece3.268

    Figure Lengend Snippet: Diagram of the portion of mitochondrial Cytochrome Oxidase I ( COI ) gene used to identify strain and the individual corn-strain (CS) haplotypes. The putative translational start site of the COI gene was arbitrarily designated as coordinate 0. Short block arrows indicate location and direction of the COI-893F and COI-1303R primers used for PCR amplification and DNA sequencing. Vertical lines within the COI gene identify polymorphic sites with the rice-strain (RS) specific EcoRV site identified. All polymorphic sites except 1287 were used to identify or confirm strain identity. The CS-h haplotypes were determined by the polymorphisms at site 1164 and 1287.

    Article Snippet: Strain identification and DNA sequence analysis In each PCR, reaction mix (30 µL) was added 5 units of the restriction enzyme EcoRV (New England Biolabs) and 4 µL of the manufacturer recommended 10× restriction enzyme buffer (final volume taken to 40 µL with water).

    Techniques: Blocking Assay, Polymerase Chain Reaction, Amplification, DNA Sequencing

    Restriction enzyme-digested fragments of the genomic DNA of A. hydrophila -phage 2. Footnote: Lane M: 1kb Plus Opti-DNA Marker (ABM, Canada); Lane L1: genomic DNA of Φ2; Lanes L2–L8: genomic DNA of Φ2 digested with EcoRV; EcoRI; Ncol; SalI; MspI; XmnI; KpnI, restriction enzymes respectively.

    Journal: Antibiotics

    Article Title: Protective Effects of Bacteriophages against Aeromonas hydrophila Causing Motile Aeromonas Septicemia (MAS) in Striped Catfish

    doi: 10.3390/antibiotics7010016

    Figure Lengend Snippet: Restriction enzyme-digested fragments of the genomic DNA of A. hydrophila -phage 2. Footnote: Lane M: 1kb Plus Opti-DNA Marker (ABM, Canada); Lane L1: genomic DNA of Φ2; Lanes L2–L8: genomic DNA of Φ2 digested with EcoRV; EcoRI; Ncol; SalI; MspI; XmnI; KpnI, restriction enzymes respectively.

    Article Snippet: The genomic DNA phages were digested using the restriction enzymes: EcoRV, EcoRI, Ncol, SalI, MspI, XmnI, and KpnI, as per the manufacturer’s instruction (ThermoFisher Scientific).

    Techniques: Marker

    Tryptase genotyping in healthy subjects. α/β-Tryptase genotyping was performed with gDNA obtained from SMCs of 24 subjects. EcoRV digestions of PCR amplimers labeled during the final cycle with DY682-labeled 3′-primer were performed. Gel images are shown for 3 of the 24 samples analyzed, 2 with a β:α ratio of 3∶1 and 1 with a ratio of 2∶2. The 17 samples in which the α-tryptase gene was detected are shown in the plot, where those with a 2∶2 or 3∶1 genotype are grouped together. Data is stored in S4 Table .

    Journal: PLoS ONE

    Article Title: A Simple, Sensitive and Safe Method to Determine the Human α/β-Tryptase Genotype

    doi: 10.1371/journal.pone.0114944

    Figure Lengend Snippet: Tryptase genotyping in healthy subjects. α/β-Tryptase genotyping was performed with gDNA obtained from SMCs of 24 subjects. EcoRV digestions of PCR amplimers labeled during the final cycle with DY682-labeled 3′-primer were performed. Gel images are shown for 3 of the 24 samples analyzed, 2 with a β:α ratio of 3∶1 and 1 with a ratio of 2∶2. The 17 samples in which the α-tryptase gene was detected are shown in the plot, where those with a 2∶2 or 3∶1 genotype are grouped together. Data is stored in S4 Table .

    Article Snippet: Reagents Betaine (B0300), Me2 SO (DMSO), glycerin, phosphate-buffered saline, 2-(N -morpholino)ethane sulfonic acid (MES), Hepes, Tris, EDTA, bovine serum albumin, agarose, and MgCl2 (Sigma-Aldrich Chemical Company LLC, St. Louis, MO); dNTP mix, gDNA purification kit, RNase-free DNase, and EcoRV (Promega Company, Fitchburg, WI); ProbeQuant G-50 Micro column (Amersham, Piscataway, NJ); FastStart Taq DNA Polymerase (Roche Diagnostics, Indianapolis, IN); polyacrylamide gels (Invitrogen, Carlsbad, CA); goat affinity-purified polyclonal IgG anti-digoxigenin (Vector Laboratories, Burlingame, CA); and IRDye680RD-labeled affinity-purified donkey IgG anti-goat IgG (LiCor, Lincoln, NE) were obtained as indicated.

    Techniques: Polymerase Chain Reaction, Labeling

    α/β-Tryptase genotyping using EcoRV-digested amplimers. EcoRV-digested amplimers were labeled with ethidium bromide after gel electrophoresis (top panels) or, during the final PCR cycle with DY682 (middle panels) or digoxigenin (bottom panels) labeled 3′-primer. gDNA from SMCs with known ββββ, βββα and ββαα genotypes, defined as 4∶0, 3∶1 and 2∶2 β:α ratios, were utilized. Experimentally calculated percentages of the β-tryptase gene were plotted against the known percentages of β-tryptase (n = 3, mean ± SD). Standard deviations were too small to visualize the error bars in the 50 and 75%β-Tryptase (theoretical) values for the middle plot. Data is stored in S2 Table .

    Journal: PLoS ONE

    Article Title: A Simple, Sensitive and Safe Method to Determine the Human α/β-Tryptase Genotype

    doi: 10.1371/journal.pone.0114944

    Figure Lengend Snippet: α/β-Tryptase genotyping using EcoRV-digested amplimers. EcoRV-digested amplimers were labeled with ethidium bromide after gel electrophoresis (top panels) or, during the final PCR cycle with DY682 (middle panels) or digoxigenin (bottom panels) labeled 3′-primer. gDNA from SMCs with known ββββ, βββα and ββαα genotypes, defined as 4∶0, 3∶1 and 2∶2 β:α ratios, were utilized. Experimentally calculated percentages of the β-tryptase gene were plotted against the known percentages of β-tryptase (n = 3, mean ± SD). Standard deviations were too small to visualize the error bars in the 50 and 75%β-Tryptase (theoretical) values for the middle plot. Data is stored in S2 Table .

    Article Snippet: Reagents Betaine (B0300), Me2 SO (DMSO), glycerin, phosphate-buffered saline, 2-(N -morpholino)ethane sulfonic acid (MES), Hepes, Tris, EDTA, bovine serum albumin, agarose, and MgCl2 (Sigma-Aldrich Chemical Company LLC, St. Louis, MO); dNTP mix, gDNA purification kit, RNase-free DNase, and EcoRV (Promega Company, Fitchburg, WI); ProbeQuant G-50 Micro column (Amersham, Piscataway, NJ); FastStart Taq DNA Polymerase (Roche Diagnostics, Indianapolis, IN); polyacrylamide gels (Invitrogen, Carlsbad, CA); goat affinity-purified polyclonal IgG anti-digoxigenin (Vector Laboratories, Burlingame, CA); and IRDye680RD-labeled affinity-purified donkey IgG anti-goat IgG (LiCor, Lincoln, NE) were obtained as indicated.

    Techniques: Labeling, Nucleic Acid Electrophoresis, Polymerase Chain Reaction

    Standard and new PCR/restriction enzyme-based methods for α and β tryptase genotyping. Genomic DNA extracts from HMC-1, Mac-6 and two SMC preparations were each tested for the presence of β and α-tryptase genes by three different methods, each using the same 5′ and 3′ PCR primer pair as described in Methods . When the 1017-28 bp amplimers were exposed to EcoRV, the α-tryptase amplimer yielded 678 and 339-40 bp fragments. After labeling with ethidium bromide, all such products were detected (upper panel). In contrast, only the high molecular weight amplimer and 339-40 bp fragment were visualized using either DY682- (middle panels) or digoxigenin- (lower panels) labeled 3′-primer. DNA sequence analyses of the restriction site are shown above the corresponding gel electrophoresis pattern.

    Journal: PLoS ONE

    Article Title: A Simple, Sensitive and Safe Method to Determine the Human α/β-Tryptase Genotype

    doi: 10.1371/journal.pone.0114944

    Figure Lengend Snippet: Standard and new PCR/restriction enzyme-based methods for α and β tryptase genotyping. Genomic DNA extracts from HMC-1, Mac-6 and two SMC preparations were each tested for the presence of β and α-tryptase genes by three different methods, each using the same 5′ and 3′ PCR primer pair as described in Methods . When the 1017-28 bp amplimers were exposed to EcoRV, the α-tryptase amplimer yielded 678 and 339-40 bp fragments. After labeling with ethidium bromide, all such products were detected (upper panel). In contrast, only the high molecular weight amplimer and 339-40 bp fragment were visualized using either DY682- (middle panels) or digoxigenin- (lower panels) labeled 3′-primer. DNA sequence analyses of the restriction site are shown above the corresponding gel electrophoresis pattern.

    Article Snippet: Reagents Betaine (B0300), Me2 SO (DMSO), glycerin, phosphate-buffered saline, 2-(N -morpholino)ethane sulfonic acid (MES), Hepes, Tris, EDTA, bovine serum albumin, agarose, and MgCl2 (Sigma-Aldrich Chemical Company LLC, St. Louis, MO); dNTP mix, gDNA purification kit, RNase-free DNase, and EcoRV (Promega Company, Fitchburg, WI); ProbeQuant G-50 Micro column (Amersham, Piscataway, NJ); FastStart Taq DNA Polymerase (Roche Diagnostics, Indianapolis, IN); polyacrylamide gels (Invitrogen, Carlsbad, CA); goat affinity-purified polyclonal IgG anti-digoxigenin (Vector Laboratories, Burlingame, CA); and IRDye680RD-labeled affinity-purified donkey IgG anti-goat IgG (LiCor, Lincoln, NE) were obtained as indicated.

    Techniques: Polymerase Chain Reaction, Labeling, Molecular Weight, Sequencing, Nucleic Acid Electrophoresis

    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:

    Restriction patterns of DNA extracted from purified virions cleaved with the selected REases. Panel A: HindIII (H), Eco32I (E32), SalI (S) and EcoRI (EI). ND, undigested DNA. M, GeneRuler 100- to 10,000-bp size marker. λ/H, λ DNA cleaved with HindIII. ~14 kb restriction fragment of EcoR32I digested virion DNA is marked with a triangle. Panel B: Arrows indicate restriction fragments assigned to ФAH14a (left) and ФAH14b (right) genomic DNA, obtained by SalI digestion.

    Journal: PLoS ONE

    Article Title: Two Inducible Prophages of an Antarctic Pseudomonas sp. ANT_H14 Use the Same Capsid for Packaging Their Genomes – Characterization of a Novel Phage Helper-Satellite System

    doi: 10.1371/journal.pone.0158889

    Figure Lengend Snippet: Restriction patterns of DNA extracted from purified virions cleaved with the selected REases. Panel A: HindIII (H), Eco32I (E32), SalI (S) and EcoRI (EI). ND, undigested DNA. M, GeneRuler 100- to 10,000-bp size marker. λ/H, λ DNA cleaved with HindIII. ~14 kb restriction fragment of EcoR32I digested virion DNA is marked with a triangle. Panel B: Arrows indicate restriction fragments assigned to ФAH14a (left) and ФAH14b (right) genomic DNA, obtained by SalI digestion.

    Article Snippet: The test for the presence of cohesive ends of the phage genome was performed as previously described [ ], using the following REases: HindIII, SalI, EcoRI, Eco32I and PstI (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Purification, Marker

    (A) AAV vector maps depicting AAV-P Tight -Cas9 and AAV-gRNA/rtTA. AAV-P Tight -Cas9 consists of a Cas9 transgene under the control of a Dox inducible Tight promoter. AAV-gRNA/rtTA consists of a gRNA expression cassette and a rtTA (Tet-On Advanced) transgene controlled by a CMV promoter. It also is designed to express GFP via an IRES element following the rtTA reading frame. (B) ICC for Cas9 and GFP was performed on 293FT cells transduced by AAV-P Tight -Cas9 and AAV-gRNA/rtTA viruses in the presence or absence of Dox. Native GFP expression is visible in virtually all of the cells (i, iii). Cas9 expression is robustly induced in the presence of Dox (ii), compared to the no Dox condition (iv). Representative images are shown. The experiment was repeated twice with similar results. (C) Diagram depicting the approximate location of where the Tet2 gRNA targets the Tet2 locus. Underlined nucleotides indicate the sequence of the Tet2 gRNA. Location of the EcoRV site and PAM sequence are denoted. (D) An approximate 460 bps region of the Tet2 locus that includes the site targeted for editing via the gRNA Tet2 , was PCR amplified from N2A genomic DNA and electrophoresed on a standard agarose gel and stained with ethidium bromide (lane 1). N2A cells were transfected with the pX330 Empty , a plasmid designed to express spCas9 and no gRNA, and 96 h later, the genomic DNA was isolated and the Tet2 locus was PCR amplified and subjected to EcoRV digestion. The PCR product was cut into two pieces of DNA as expected (lane 2). However, when N2A cells were transfected with pX330 Tet2 and similarly processed, the PCR product was incompletely digested resulting in a total of three bands on the gel - one uncut PCR product (~460 bps) and two smaller bands. In this case the genome editing was ~33%. (E) Edited DNA depicted in ( D , lane 3) was gel purified and TA cloned and 6 independent clones were sequenced. These 6 clones contained deletions which destroyed the EcoRV site.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing

    doi: 10.3389/fnmol.2016.00070

    Figure Lengend Snippet: (A) AAV vector maps depicting AAV-P Tight -Cas9 and AAV-gRNA/rtTA. AAV-P Tight -Cas9 consists of a Cas9 transgene under the control of a Dox inducible Tight promoter. AAV-gRNA/rtTA consists of a gRNA expression cassette and a rtTA (Tet-On Advanced) transgene controlled by a CMV promoter. It also is designed to express GFP via an IRES element following the rtTA reading frame. (B) ICC for Cas9 and GFP was performed on 293FT cells transduced by AAV-P Tight -Cas9 and AAV-gRNA/rtTA viruses in the presence or absence of Dox. Native GFP expression is visible in virtually all of the cells (i, iii). Cas9 expression is robustly induced in the presence of Dox (ii), compared to the no Dox condition (iv). Representative images are shown. The experiment was repeated twice with similar results. (C) Diagram depicting the approximate location of where the Tet2 gRNA targets the Tet2 locus. Underlined nucleotides indicate the sequence of the Tet2 gRNA. Location of the EcoRV site and PAM sequence are denoted. (D) An approximate 460 bps region of the Tet2 locus that includes the site targeted for editing via the gRNA Tet2 , was PCR amplified from N2A genomic DNA and electrophoresed on a standard agarose gel and stained with ethidium bromide (lane 1). N2A cells were transfected with the pX330 Empty , a plasmid designed to express spCas9 and no gRNA, and 96 h later, the genomic DNA was isolated and the Tet2 locus was PCR amplified and subjected to EcoRV digestion. The PCR product was cut into two pieces of DNA as expected (lane 2). However, when N2A cells were transfected with pX330 Tet2 and similarly processed, the PCR product was incompletely digested resulting in a total of three bands on the gel - one uncut PCR product (~460 bps) and two smaller bands. In this case the genome editing was ~33%. (E) Edited DNA depicted in ( D , lane 3) was gel purified and TA cloned and 6 independent clones were sequenced. These 6 clones contained deletions which destroyed the EcoRV site.

    Article Snippet: Three microliters of PCR product was digested with 5 units of EcoRV-HF (New England Biolabs) for 2 h in a standard 10 μl restriction enzyme reaction following the manufacturer's instructions.

    Techniques: Plasmid Preparation, Expressing, Immunocytochemistry, Sequencing, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining, Transfection, Isolation, Purification, Clone Assay

    Isolation of genetically corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cells Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green, 1987 ) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm. Quantitative RT–PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32 P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.

    Journal: EMBO Molecular Medicine

    Article Title: A single epidermal stem cell strategy for safe ex vivo gene therapy

    doi: 10.15252/emmm.201404353

    Figure Lengend Snippet: Isolation of genetically corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cells Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green, 1987 ) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm. Quantitative RT–PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32 P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.

    Article Snippet: Ten micrograms of DNA was codigested with SpeI/EcoRV HF (New England Biolabs) and loaded on a 0.8% agarose (Promega) gel.

    Techniques: Isolation, Infection, Clone Assay, Immunostaining, Expressing, Staining, Irradiation, Quantitative RT-PCR, Southern Blot, Western Blot, Negative Control, Positive Control

    Full-length wild-type p53 binds to MYC G-quadruplex more efficiently than its isolated C-terminal and central regions The role of p53 domains in MYC G-quadruplex binding was studied by EMSA. Oligonucleotides (1 pmol) Pu52 ( A ), Pu33 ( B ) and Pu22 ( C ) were incubated with wtp53 (lanes 2–5; 50, 100, 200, 400 ng), p53-320 (lanes 6–9; 25, 50, 100, 200 ng) or p53CD (lanes 10–13; 12.5, 25, 50, 100 ng) in the presence of 10 ng of non-specific competitor pBSK/EcoRV. Binding of full-length wtp53, central DBD and C-terminal construct of p53 to double-stranded oligonucleotides containing p53CON was studied by EMSA. Oligonucleotides (0.25 pmol) ( D ) P1-50, ( E ) P1-30 and ( F ) P1-22 were incubated with wtp53 (lanes 2–5; 12.5, 25, 50, 100 ng), p53-320 (lanes 6–9; 6, 12.5, 25, 50 ng) or p53CD (lanes 10–13; 3, 6, 12.5, 25 ng) in the presence of 2.5 ng of non-specific competitor pBSK/EcoRV.

    Journal: Bioscience Reports

    Article Title: Wild-type p53 binds to MYC promoter G-quadruplex

    doi: 10.1042/BSR20160232

    Figure Lengend Snippet: Full-length wild-type p53 binds to MYC G-quadruplex more efficiently than its isolated C-terminal and central regions The role of p53 domains in MYC G-quadruplex binding was studied by EMSA. Oligonucleotides (1 pmol) Pu52 ( A ), Pu33 ( B ) and Pu22 ( C ) were incubated with wtp53 (lanes 2–5; 50, 100, 200, 400 ng), p53-320 (lanes 6–9; 25, 50, 100, 200 ng) or p53CD (lanes 10–13; 12.5, 25, 50, 100 ng) in the presence of 10 ng of non-specific competitor pBSK/EcoRV. Binding of full-length wtp53, central DBD and C-terminal construct of p53 to double-stranded oligonucleotides containing p53CON was studied by EMSA. Oligonucleotides (0.25 pmol) ( D ) P1-50, ( E ) P1-30 and ( F ) P1-22 were incubated with wtp53 (lanes 2–5; 12.5, 25, 50, 100 ng), p53-320 (lanes 6–9; 6, 12.5, 25, 50 ng) or p53CD (lanes 10–13; 3, 6, 12.5, 25 ng) in the presence of 2.5 ng of non-specific competitor pBSK/EcoRV.

    Article Snippet: Non-specific competitor plasmid pBSK/EcoRV was prepared by EcoRV restriction endonuclease (New England Biolabs) cleavage of pBSK.

    Techniques: Isolation, Binding Assay, Incubation, Construct

    Full-length wild-type p53 binding to the parallel G-quadruplex from MYC promoter NHE III 1 region is comparable with p53CON binding ( A ) Comparison of sequence-specific and MYC G-quadruplex Pu52 binding of p53 by EMSA. Oligonucelotides P1-50 (0.25 pmol, lanes 1–5), Pu52 (1 pmol, lanes 6–10) and A50 (0.25 pmol, lanes 11–15) were incubated with wtp53 protein (50, 100, 200, 400 ng/1 pmol of oligonucleotide) in the presence of 20 ng (per 1 pmol of oligonucleotide) of non-specific competitor pBSK/EcoRV. ( B ) CD spectra of Pu52 oligonucleotide measured in 5 mM Tris pH 7.6 (dotted line) and after addition of 10 mM KCl (dashed line) and 50 mM KCl respectively (solid line). ( C ) DMS footprinting of Pu52 oligonucleotide. Pu52 was annealed in 50 mM KCl without subsequent DMS treatment (lane 1), annealed in the absence of KCl and treated with DMS (lane 2) or annealed in 50 mM KCl and treated with DMS (lane 3). ( D ) Comparison of sequence-specific and MYC G-quadruplex Pu22 binding of p53 by EMSA. Oligonucelotides P1-22 (0.25 pmol, lanes 1–5), Pu22 (1 pmol, lanes 6–10) and A25 (0.25 pmol, lanes 11–15) were incubated with wtp53 protein (50, 100, 200, 400 ng/1 pmol of oligonucleotide) in the presence of 20 ng (per 1 pmol of oligonucleotide) of non-specific competitor pBSK/EcoRV. ( E ) CD spectra of Pu22 oligonucleotide measured in 5 mM Tris pH 7.6 (dotted line) and after addition of 10 mM KCl (dashed line) and 50 mM KCl respectively (solid line). ( F ) DMS footprinting of Pu22 oligonucleotide. Pu22 was annealed in 50 mM KCl without subsequent DMS treatment (lane 1), annealed in the absence of KCl and treated with DMS (lane 2) or annealed in 50 mM KCl and treated with DMS (lane 3).

    Journal: Bioscience Reports

    Article Title: Wild-type p53 binds to MYC promoter G-quadruplex

    doi: 10.1042/BSR20160232

    Figure Lengend Snippet: Full-length wild-type p53 binding to the parallel G-quadruplex from MYC promoter NHE III 1 region is comparable with p53CON binding ( A ) Comparison of sequence-specific and MYC G-quadruplex Pu52 binding of p53 by EMSA. Oligonucelotides P1-50 (0.25 pmol, lanes 1–5), Pu52 (1 pmol, lanes 6–10) and A50 (0.25 pmol, lanes 11–15) were incubated with wtp53 protein (50, 100, 200, 400 ng/1 pmol of oligonucleotide) in the presence of 20 ng (per 1 pmol of oligonucleotide) of non-specific competitor pBSK/EcoRV. ( B ) CD spectra of Pu52 oligonucleotide measured in 5 mM Tris pH 7.6 (dotted line) and after addition of 10 mM KCl (dashed line) and 50 mM KCl respectively (solid line). ( C ) DMS footprinting of Pu52 oligonucleotide. Pu52 was annealed in 50 mM KCl without subsequent DMS treatment (lane 1), annealed in the absence of KCl and treated with DMS (lane 2) or annealed in 50 mM KCl and treated with DMS (lane 3). ( D ) Comparison of sequence-specific and MYC G-quadruplex Pu22 binding of p53 by EMSA. Oligonucelotides P1-22 (0.25 pmol, lanes 1–5), Pu22 (1 pmol, lanes 6–10) and A25 (0.25 pmol, lanes 11–15) were incubated with wtp53 protein (50, 100, 200, 400 ng/1 pmol of oligonucleotide) in the presence of 20 ng (per 1 pmol of oligonucleotide) of non-specific competitor pBSK/EcoRV. ( E ) CD spectra of Pu22 oligonucleotide measured in 5 mM Tris pH 7.6 (dotted line) and after addition of 10 mM KCl (dashed line) and 50 mM KCl respectively (solid line). ( F ) DMS footprinting of Pu22 oligonucleotide. Pu22 was annealed in 50 mM KCl without subsequent DMS treatment (lane 1), annealed in the absence of KCl and treated with DMS (lane 2) or annealed in 50 mM KCl and treated with DMS (lane 3).

    Article Snippet: Non-specific competitor plasmid pBSK/EcoRV was prepared by EcoRV restriction endonuclease (New England Biolabs) cleavage of pBSK.

    Techniques: Binding Assay, Sequencing, Incubation, Footprinting

    Wild-type p53 and C-terminal region of p53 bind G-quadruplex Pu52 with higher affinity than double-stranded Pu52/Py52 Binding of various p53 protein constructs to MYC promoter G-quadruplexes from was studied by EMSA. Oligonucleotides ( A ) P1-40 (0.5 pmol), ( B ) Pu52 (1 pmol) and ( C ) dsPu52/Py52 (0.5 pmol) were incubated with wtp53 (lanes 2–4; 25, 50, 100 ng/1 pmol of oligonucleotide), CΔ30 (lanes 5–7; 25, 50, 100 ng/1 pmol of oligonucleotide), p53CT (lanes 8–10; 50, 100, 200 ng/1 pmol of oligonucleotide), p53T (lanes 11–13; 50, 100, 200 ng/1 pmol of oligonucleotide) or p53CD (lanes 14 and 15; 100, 200 ng/1 pmol of oligonucleotide) in the presence of 20 ng (per 1 pmol of oligonucleotide) of non-specific competitor pBSK/EcoRV.

    Journal: Bioscience Reports

    Article Title: Wild-type p53 binds to MYC promoter G-quadruplex

    doi: 10.1042/BSR20160232

    Figure Lengend Snippet: Wild-type p53 and C-terminal region of p53 bind G-quadruplex Pu52 with higher affinity than double-stranded Pu52/Py52 Binding of various p53 protein constructs to MYC promoter G-quadruplexes from was studied by EMSA. Oligonucleotides ( A ) P1-40 (0.5 pmol), ( B ) Pu52 (1 pmol) and ( C ) dsPu52/Py52 (0.5 pmol) were incubated with wtp53 (lanes 2–4; 25, 50, 100 ng/1 pmol of oligonucleotide), CΔ30 (lanes 5–7; 25, 50, 100 ng/1 pmol of oligonucleotide), p53CT (lanes 8–10; 50, 100, 200 ng/1 pmol of oligonucleotide), p53T (lanes 11–13; 50, 100, 200 ng/1 pmol of oligonucleotide) or p53CD (lanes 14 and 15; 100, 200 ng/1 pmol of oligonucleotide) in the presence of 20 ng (per 1 pmol of oligonucleotide) of non-specific competitor pBSK/EcoRV.

    Article Snippet: Non-specific competitor plasmid pBSK/EcoRV was prepared by EcoRV restriction endonuclease (New England Biolabs) cleavage of pBSK.

    Techniques: Binding Assay, Construct, Incubation