puc19  (New England Biolabs)


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    Name:
    pUC19 Vector
    Description:
    pUC19 Vector 250 ug
    Catalog Number:
    N3041L
    Price:
    300
    Category:
    Vectors Plasmids
    Size:
    250 ug
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    Structured Review

    New England Biolabs puc19
    pUC19 Vector
    pUC19 Vector 250 ug
    https://www.bioz.com/result/puc19/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    puc19 - by Bioz Stars, 2021-07
    86/100 stars

    Images

    1) Product Images from "Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR–Cas9 variants"

    Article Title: Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR–Cas9 variants

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab163

    Cas9 variants have different cleavage activities against mismatched targets. ( A ) Representative agarose gels showing cleavage of a negatively supercoiled (nSC) plasmid containing the perfect target (0 MM) or mismatched (2 to 5 MM) target over a time course by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 15 s, 30 s, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h and 5 h. tr:crRNA = tracrRNA:crRNA. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (–) = pTarget or pLibrary alone incubated at 37°C for the longest time point in the assay (5 h); (-cr) = pTarget or pLibrary incubated with Cas9 only at 37°C for the longest time point in the assay (5 h); n = Nt.BspQI nicked pUC19; li = BsaI-HF linearized pUC19. ( B ) Quantification of supercoiled, linear and nicked pools from cleavage of perfect or fully crRNA-complementary (0 MM) and mismatched (2 to 5 MM) target plasmid by Cas9 after 10 min and 3 h. pTarget MM indicates target plasmid (0, 2 to 5 MM) alone incubated at 37°C for the time points indicated. Target sequences tested are listed with PAM (bold) and mismatches (lowercase and red) indicated. (–) indicates a cleavage reaction with the target plasmid and Cas9 only, and (+) indicates a cleavage reaction with the target plasmid, Cas9 and cognate tracrRNA:crRNA. Values plotted represent an average of three replicates. Error bars are SEM. * or • indicate P
    Figure Legend Snippet: Cas9 variants have different cleavage activities against mismatched targets. ( A ) Representative agarose gels showing cleavage of a negatively supercoiled (nSC) plasmid containing the perfect target (0 MM) or mismatched (2 to 5 MM) target over a time course by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 15 s, 30 s, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h and 5 h. tr:crRNA = tracrRNA:crRNA. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (–) = pTarget or pLibrary alone incubated at 37°C for the longest time point in the assay (5 h); (-cr) = pTarget or pLibrary incubated with Cas9 only at 37°C for the longest time point in the assay (5 h); n = Nt.BspQI nicked pUC19; li = BsaI-HF linearized pUC19. ( B ) Quantification of supercoiled, linear and nicked pools from cleavage of perfect or fully crRNA-complementary (0 MM) and mismatched (2 to 5 MM) target plasmid by Cas9 after 10 min and 3 h. pTarget MM indicates target plasmid (0, 2 to 5 MM) alone incubated at 37°C for the time points indicated. Target sequences tested are listed with PAM (bold) and mismatches (lowercase and red) indicated. (–) indicates a cleavage reaction with the target plasmid and Cas9 only, and (+) indicates a cleavage reaction with the target plasmid, Cas9 and cognate tracrRNA:crRNA. Values plotted represent an average of three replicates. Error bars are SEM. * or • indicate P

    Techniques Used: Plasmid Preparation, Incubation

    2) Product Images from "Relationship of DNA degradation by Saccharomyces cerevisiae Exonuclease 1 and its stimulation by RPA and Mre11-Rad50-Xrs2 to DNA end resection"

    Article Title: Relationship of DNA degradation by Saccharomyces cerevisiae Exonuclease 1 and its stimulation by RPA and Mre11-Rad50-Xrs2 to DNA end resection

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1305166110

    Exo1 does not stimulate DNA end resection by Dna2, Sgs1, and Top3-Rmi1. ( A ) Nuclease assays with Exo1 (7 nM), Dna2 (7 nM), Sgs1 (7 nM), Top3-Rmi1 (14 nM), and unlabeled pUC19 dsDNA containing 4-nt ssDNA overhangs at the 5′ ends (3.8 nM) in the presence of RPA (3 μM) for 4, 8, or 12 min. The gel is an inverted image of ethidium bromide-stained DNA. ( B ) Quantification of experiments as shown in A . Error bars show SE. ( C ) Nuclease assays with Exo1 (1 nM), Sgs1 (1 nM), Top3-Rmi1 (3 nM), Dna2 (1 nM), Mre11-Rad50-Xrs2 (25 nM), as indicated, for 1, 2, 4, 8, and 16 min in the presence of RPA (0.4 μM). Blunt-ended pUC19 dsDNA (1 nM), 32 P labeled at the 3′ end, was used. ( D ) Quantification of experiments as shown in C . Error bars show SE.
    Figure Legend Snippet: Exo1 does not stimulate DNA end resection by Dna2, Sgs1, and Top3-Rmi1. ( A ) Nuclease assays with Exo1 (7 nM), Dna2 (7 nM), Sgs1 (7 nM), Top3-Rmi1 (14 nM), and unlabeled pUC19 dsDNA containing 4-nt ssDNA overhangs at the 5′ ends (3.8 nM) in the presence of RPA (3 μM) for 4, 8, or 12 min. The gel is an inverted image of ethidium bromide-stained DNA. ( B ) Quantification of experiments as shown in A . Error bars show SE. ( C ) Nuclease assays with Exo1 (1 nM), Sgs1 (1 nM), Top3-Rmi1 (3 nM), Dna2 (1 nM), Mre11-Rad50-Xrs2 (25 nM), as indicated, for 1, 2, 4, 8, and 16 min in the presence of RPA (0.4 μM). Blunt-ended pUC19 dsDNA (1 nM), 32 P labeled at the 3′ end, was used. ( D ) Quantification of experiments as shown in C . Error bars show SE.

    Techniques Used: Recombinase Polymerase Amplification, Staining, Labeling

    Sgs1 does not stimulate resection of dsDNA by Exo1. ( A ) Nuclease assays with Exo1 (0.35, 0.53, 0.8, 1.2, and 1.8 nM), RPA (0.4 μM), and either without (lanes 2–6) or with Sgs1 (0.1 nM, lanes 8–13) in low-salt buffer. Blunt-ended pUC19 dsDNA (1 nM), 32 P labeled at the 3′ end, was used. ( B ) Quantification of experiments as shown in A . Error bars show SE. ( C ) Nuclease assays with Exo1 (0.53, 0.8, 1.2, 1.8, and 2.7 nM), RPA (0.4 μM), and either without (lanes 2–6) or with Sgs1 (0.5 nM) and Top3-Rmi1 (5 nM, lanes 9–14, respectively), in standard buffer. Substrate is as in A . ( D ) Quantification of experiments as shown in C . Error bars show SE. ( E ) Nuclease assay carried out with Exo1 (0.5, 1, 2, 3, and 4 nM), RPA (0.4 μM), and either without (lanes 2–6) or with helicase-dead Sgs1 K706A (20 nM, lanes 8–12). Substrate is as in A . ( F ) Increasing amounts of nuclease-dead Exo1 D173A (0.53, 0.8, 1.2, 1.8, 2.7, 4, and 8 nM) were added to reactions containing Sgs1 (0.5 nM) and/or Top3-Rmi1 (5 nM), as indicated, in the presence of RPA (0.4 μM). Substrate is as in A .
    Figure Legend Snippet: Sgs1 does not stimulate resection of dsDNA by Exo1. ( A ) Nuclease assays with Exo1 (0.35, 0.53, 0.8, 1.2, and 1.8 nM), RPA (0.4 μM), and either without (lanes 2–6) or with Sgs1 (0.1 nM, lanes 8–13) in low-salt buffer. Blunt-ended pUC19 dsDNA (1 nM), 32 P labeled at the 3′ end, was used. ( B ) Quantification of experiments as shown in A . Error bars show SE. ( C ) Nuclease assays with Exo1 (0.53, 0.8, 1.2, 1.8, and 2.7 nM), RPA (0.4 μM), and either without (lanes 2–6) or with Sgs1 (0.5 nM) and Top3-Rmi1 (5 nM, lanes 9–14, respectively), in standard buffer. Substrate is as in A . ( D ) Quantification of experiments as shown in C . Error bars show SE. ( E ) Nuclease assay carried out with Exo1 (0.5, 1, 2, 3, and 4 nM), RPA (0.4 μM), and either without (lanes 2–6) or with helicase-dead Sgs1 K706A (20 nM, lanes 8–12). Substrate is as in A . ( F ) Increasing amounts of nuclease-dead Exo1 D173A (0.53, 0.8, 1.2, 1.8, 2.7, 4, and 8 nM) were added to reactions containing Sgs1 (0.5 nM) and/or Top3-Rmi1 (5 nM), as indicated, in the presence of RPA (0.4 μM). Substrate is as in A .

    Techniques Used: Recombinase Polymerase Amplification, Labeling, Nuclease Assay

    In the presence of RPA, resection of linear plasmid-length dsDNA by Exo1 produces kilobase-sized ssDNA. ( A ) Nuclease activity of Exo1 (2.7 nM) as a function of time (0.5, 1, 2, 4, 6, 10, 15, 20, and 30 min). pUC19 dsDNA (blunt, 1 nM), 32 P labeled at the 3′ end, was the substrate. “Heat” refers to ssDNA produced by heat denaturation of the pUC19 dsDNA. ( B ) Reaction as in A carried out in the presence of RPA (0.4 μM). ( C ) Illustration summarizing results from A and B , showing the intermediates and products of dsDNA degradation by Exo1 in the presence or absence of RPA.
    Figure Legend Snippet: In the presence of RPA, resection of linear plasmid-length dsDNA by Exo1 produces kilobase-sized ssDNA. ( A ) Nuclease activity of Exo1 (2.7 nM) as a function of time (0.5, 1, 2, 4, 6, 10, 15, 20, and 30 min). pUC19 dsDNA (blunt, 1 nM), 32 P labeled at the 3′ end, was the substrate. “Heat” refers to ssDNA produced by heat denaturation of the pUC19 dsDNA. ( B ) Reaction as in A carried out in the presence of RPA (0.4 μM). ( C ) Illustration summarizing results from A and B , showing the intermediates and products of dsDNA degradation by Exo1 in the presence or absence of RPA.

    Techniques Used: Recombinase Polymerase Amplification, Plasmid Preparation, Activity Assay, Labeling, Produced

    Mre11-Rad50-Xrs2 complex stimulates resection of dsDNA by Exo1. ( A ) A representative experiment with blunt-ended pUC19 dsDNA (1 nM, 32 P labeled at the 3′ end) showing degradation by Exo1 (0.4 nM, where indicated) and its stimulation by MRX [1, 3, 10, 30, and 100 nM (lanes 2–6) and 1, 3, 10, and 30 nM (lanes 8–11), respectively]. ( B ) Reaction as in A carried out in the presence of RPA (0.4 μM). ( C ) Quantitation of experiments as in A and B . Error bars show SE.
    Figure Legend Snippet: Mre11-Rad50-Xrs2 complex stimulates resection of dsDNA by Exo1. ( A ) A representative experiment with blunt-ended pUC19 dsDNA (1 nM, 32 P labeled at the 3′ end) showing degradation by Exo1 (0.4 nM, where indicated) and its stimulation by MRX [1, 3, 10, 30, and 100 nM (lanes 2–6) and 1, 3, 10, and 30 nM (lanes 8–11), respectively]. ( B ) Reaction as in A carried out in the presence of RPA (0.4 μM). ( C ) Quantitation of experiments as in A and B . Error bars show SE.

    Techniques Used: Labeling, Recombinase Polymerase Amplification, Quantitation Assay

    Exo1 preferentially degrades dsDNA resected at the 5′ end to produce an ssDNA tail at the 3′ end. ( A ) Quantification of Exo1 nuclease activity, in the presence of RPA (3 μM), using unlabeled pUC19 dsDNA (7.6 nM) that either was blunt or contained an ssDNA overhang (4 nt) at either 3′ or 5′ ends. Error bars indicate SE. ( B ) A representative experiment showing degradation of pUC19 dsDNA with a 5′-ssDNA overhang of 3 nt (7 nM, 32 P labeled at the 3′ end) by Exo1 (0.05, 0.15, 0.45, 1.3, 4, and 12 nM, respectively) in the presence of RPA (2.8 μM). “D173A”: The nuclease-deficient Exo1 D173A mutant was used instead of wild-type Exo1 (12 nM). “Heat”: Heat-denatured substrate. ( C ) Illustration summarizing results from B showing degradation by Exo1 of a dsDNA substrate with 5′-end ssDNA overhangs. ( D ) Quantification of Exo1 nuclease activity on 3.0 kb unlabeled dsDNA (7.6 nM) containing an ssDNA overhang of 4 nt at both 5′ ends (squares) vs. dsDNA with a 4-nt 5′ overhang at one end and a 3′ (circles) or 5′ (triangles) overhang of ∼100 nt at the other end. The reactions were carried out in the presence of RPA (3 μM). Error bars show SE.
    Figure Legend Snippet: Exo1 preferentially degrades dsDNA resected at the 5′ end to produce an ssDNA tail at the 3′ end. ( A ) Quantification of Exo1 nuclease activity, in the presence of RPA (3 μM), using unlabeled pUC19 dsDNA (7.6 nM) that either was blunt or contained an ssDNA overhang (4 nt) at either 3′ or 5′ ends. Error bars indicate SE. ( B ) A representative experiment showing degradation of pUC19 dsDNA with a 5′-ssDNA overhang of 3 nt (7 nM, 32 P labeled at the 3′ end) by Exo1 (0.05, 0.15, 0.45, 1.3, 4, and 12 nM, respectively) in the presence of RPA (2.8 μM). “D173A”: The nuclease-deficient Exo1 D173A mutant was used instead of wild-type Exo1 (12 nM). “Heat”: Heat-denatured substrate. ( C ) Illustration summarizing results from B showing degradation by Exo1 of a dsDNA substrate with 5′-end ssDNA overhangs. ( D ) Quantification of Exo1 nuclease activity on 3.0 kb unlabeled dsDNA (7.6 nM) containing an ssDNA overhang of 4 nt at both 5′ ends (squares) vs. dsDNA with a 4-nt 5′ overhang at one end and a 3′ (circles) or 5′ (triangles) overhang of ∼100 nt at the other end. The reactions were carried out in the presence of RPA (3 μM). Error bars show SE.

    Techniques Used: Activity Assay, Recombinase Polymerase Amplification, Labeling, Mutagenesis

    3) Product Images from "Nucleosome Spacing Generated by ISWI and CHD1 Remodelers Is Constant Regardless of Nucleosome Density"

    Article Title: Nucleosome Spacing Generated by ISWI and CHD1 Remodelers Is Constant Regardless of Nucleosome Density

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01070-14

    ISWI, ACF, and Chd1 have clamping activity, i.e., they generate very similar and constant nucleosomal repeat lengths regardless of nucleosome density. (A) Limited digests with the indicated MNase concentrations of SGD chromatin (plasmid pUC19-PHO8) at the indicated assembly degrees after incubation with (+ISWI) or without (−ISWI) ISWI remodeler. Asterisks denote the trinucleosomal fragment band. MNase digestion fragments were visualized by Southern blotting and probing against the yeast PHO8 gene. M, DNA marker (2-log; NEB). (B and C) As for panel A but for the ACF or Chd1 remodeler, respectively.
    Figure Legend Snippet: ISWI, ACF, and Chd1 have clamping activity, i.e., they generate very similar and constant nucleosomal repeat lengths regardless of nucleosome density. (A) Limited digests with the indicated MNase concentrations of SGD chromatin (plasmid pUC19-PHO8) at the indicated assembly degrees after incubation with (+ISWI) or without (−ISWI) ISWI remodeler. Asterisks denote the trinucleosomal fragment band. MNase digestion fragments were visualized by Southern blotting and probing against the yeast PHO8 gene. M, DNA marker (2-log; NEB). (B and C) As for panel A but for the ACF or Chd1 remodeler, respectively.

    Techniques Used: Activity Assay, Plasmid Preparation, Incubation, Southern Blot, Marker

    4) Product Images from "Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974 ▿"

    Article Title: Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974 ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00782-09

    In vivo activities of Streptomyces SerRS1 and SerRS2. s erS1 , s erS2 , E. coli s erS , and the s erS2 ( H270G ) mutant were cloned into pUC19 (pUC) vector under the control of the glnS ). (A) The plasmids were transformed into an E. coli temperature-sensitive
    Figure Legend Snippet: In vivo activities of Streptomyces SerRS1 and SerRS2. s erS1 , s erS2 , E. coli s erS , and the s erS2 ( H270G ) mutant were cloned into pUC19 (pUC) vector under the control of the glnS ). (A) The plasmids were transformed into an E. coli temperature-sensitive

    Techniques Used: In Vivo, Mutagenesis, Clone Assay, Plasmid Preparation, Transformation Assay

    5) Product Images from "Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974 ▿"

    Article Title: Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974 ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00782-09

    In vivo activities of Streptomyces SerRS1 and SerRS2. s erS1 , s erS2 , E. coli s erS , and the s erS2 ( H270G ) mutant were cloned into pUC19 (pUC) vector under the control of the glnS ). (A) The plasmids were transformed into an E. coli temperature-sensitive
    Figure Legend Snippet: In vivo activities of Streptomyces SerRS1 and SerRS2. s erS1 , s erS2 , E. coli s erS , and the s erS2 ( H270G ) mutant were cloned into pUC19 (pUC) vector under the control of the glnS ). (A) The plasmids were transformed into an E. coli temperature-sensitive

    Techniques Used: In Vivo, Mutagenesis, Clone Assay, Plasmid Preparation, Transformation Assay

    6) Product Images from "Secretory expression of biologically active human Herpes virus interleukin-10 analogues in Escherichia coli via a modified Sec-dependent transporter construct"

    Article Title: Secretory expression of biologically active human Herpes virus interleukin-10 analogues in Escherichia coli via a modified Sec-dependent transporter construct

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-13-82

    Immunoblot analysis of viral IL-10 recombinant proteins. E. coli BL21 (DE3) derived recombinant viral IL-10 proteins were analyzed by immunoblot in different concentrations and from different cell compartments under both reducing ( A = HCMV IL-10; C = EBV IL-10) and non-reducing conditions ( B = HCMV IL-10). Periplasmic and cytoplasmic fractions of pUC19 transformed E. coli BL21 (DE3) cells and commercial viral IL-10 proteins served as controls. One experiment representative of three is shown. SN = supernatant; PP = periplasmic fraction; CP = cytoplasmic fraction; Com. = commercial; Rec. = recombinant.
    Figure Legend Snippet: Immunoblot analysis of viral IL-10 recombinant proteins. E. coli BL21 (DE3) derived recombinant viral IL-10 proteins were analyzed by immunoblot in different concentrations and from different cell compartments under both reducing ( A = HCMV IL-10; C = EBV IL-10) and non-reducing conditions ( B = HCMV IL-10). Periplasmic and cytoplasmic fractions of pUC19 transformed E. coli BL21 (DE3) cells and commercial viral IL-10 proteins served as controls. One experiment representative of three is shown. SN = supernatant; PP = periplasmic fraction; CP = cytoplasmic fraction; Com. = commercial; Rec. = recombinant.

    Techniques Used: Recombinant, Derivative Assay, Transformation Assay

    Plasmid maps of HCMV IL-10 (pAZ1c; A) and EBV (pGA6; B) expression vectors are depicted. The artificial transporter consists of the E. coli ompF signal sequence fused in frame to E. coli codon optimized mature viral IL-10 genes under control of the T7 promoter. For subcloning, the constructs are flanked by Eco RI restriction sites. Plasmid pGA6 contains a ColE1, pAZ1c a pUC19-derived pMB1 origin of replication.
    Figure Legend Snippet: Plasmid maps of HCMV IL-10 (pAZ1c; A) and EBV (pGA6; B) expression vectors are depicted. The artificial transporter consists of the E. coli ompF signal sequence fused in frame to E. coli codon optimized mature viral IL-10 genes under control of the T7 promoter. For subcloning, the constructs are flanked by Eco RI restriction sites. Plasmid pGA6 contains a ColE1, pAZ1c a pUC19-derived pMB1 origin of replication.

    Techniques Used: Plasmid Preparation, Expressing, Sequencing, Subcloning, Construct, Derivative Assay

    Activation of STAT3 by E. coli derived viral IL-10. STAT3 phosphorylation (STAT3-pY705) was analyzed by immunoblot of protein extracts from human Daudi cells (HCMV IL-10; A) and J774.1 mouse macrophages (EBV IL-10; B) treated with different concentrations of bacteria-derived viral IL-10. Total STAT3 was used to ensure equal protein loading in all lanes (double bands in Daudi cells represent STAT3 isoforms α and β). Periplasmic and cytoplasmic fractions of pUC19 transformed E. coli BL21 (DE3) cells and commercial viral IL-10 proteins served as controls. One experiment representative of two is shown. PP = periplasmic fraction; CP = cytoplasmic fraction; Com. = commercial; Rec. = recombinant.
    Figure Legend Snippet: Activation of STAT3 by E. coli derived viral IL-10. STAT3 phosphorylation (STAT3-pY705) was analyzed by immunoblot of protein extracts from human Daudi cells (HCMV IL-10; A) and J774.1 mouse macrophages (EBV IL-10; B) treated with different concentrations of bacteria-derived viral IL-10. Total STAT3 was used to ensure equal protein loading in all lanes (double bands in Daudi cells represent STAT3 isoforms α and β). Periplasmic and cytoplasmic fractions of pUC19 transformed E. coli BL21 (DE3) cells and commercial viral IL-10 proteins served as controls. One experiment representative of two is shown. PP = periplasmic fraction; CP = cytoplasmic fraction; Com. = commercial; Rec. = recombinant.

    Techniques Used: Activation Assay, Derivative Assay, Transformation Assay, Recombinant

    Inhibition of LPS-induced TNF-α release by E. coli derived recombinant EBV IL-10. J774.1 mouse macrophages were incubated with E. coli BL21 (DE3) pGA6 periplasmic fraction alone (bacterial recombinant EBV IL-10 at ~ 400 ng/ml) or in the presence of neutralizing monoclonal anti-EBV IL-10 antibody. Periplasmic fractions of E. coli BL21 (DE3) pUC19 were used as TNF-α induction control. Commercial EBV IL-10 (at ~ 400 ng/ml) served as positive control. TNF-α induction levels were set at 100%, and changes of TNF-α release are the means ± SD of four independent experiments. Statistical significance was determined using the Student t-test. Asterisks indicate statistically significant differences (* p ≤ 0.05; ** p ≤ 0.01) between pGA6 PP, pUC19 PP, and pGA6 after anti-EBV IL-10 treatment. PP = periplasmic fraction; Com. = commercial; Rec. = recombinant; mAb = monoclonal antibody.
    Figure Legend Snippet: Inhibition of LPS-induced TNF-α release by E. coli derived recombinant EBV IL-10. J774.1 mouse macrophages were incubated with E. coli BL21 (DE3) pGA6 periplasmic fraction alone (bacterial recombinant EBV IL-10 at ~ 400 ng/ml) or in the presence of neutralizing monoclonal anti-EBV IL-10 antibody. Periplasmic fractions of E. coli BL21 (DE3) pUC19 were used as TNF-α induction control. Commercial EBV IL-10 (at ~ 400 ng/ml) served as positive control. TNF-α induction levels were set at 100%, and changes of TNF-α release are the means ± SD of four independent experiments. Statistical significance was determined using the Student t-test. Asterisks indicate statistically significant differences (* p ≤ 0.05; ** p ≤ 0.01) between pGA6 PP, pUC19 PP, and pGA6 after anti-EBV IL-10 treatment. PP = periplasmic fraction; Com. = commercial; Rec. = recombinant; mAb = monoclonal antibody.

    Techniques Used: Inhibition, Derivative Assay, Recombinant, Incubation, Positive Control

    7) Product Images from "C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents"

    Article Title: C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1097

    ( A ) Influence of ionic strength on pUC19 condensation (400 ng) by OC3 and MC3 (25 μM) opioid compounds. Condensation reactions on pUC19 (400 ng) by opioid compounds in ( B ) acidic NaOAc buffer (80 mM, pH = 4.0), and ( C ) basic Tris buffer (80 mM, pH = 9.0) in the presence of 25 mM NaCl.
    Figure Legend Snippet: ( A ) Influence of ionic strength on pUC19 condensation (400 ng) by OC3 and MC3 (25 μM) opioid compounds. Condensation reactions on pUC19 (400 ng) by opioid compounds in ( B ) acidic NaOAc buffer (80 mM, pH = 4.0), and ( C ) basic Tris buffer (80 mM, pH = 9.0) in the presence of 25 mM NaCl.

    Techniques Used:

    ( A ) Agarose gel electrophoresis of supercoiled (400 ng) and ( B ) a 742 bp dsDNA fragment of pUC19 (400 ng) exposed to increasing concentrations of MC3, OC3 and HC3 . Reactions were carried out in the presence of 25 mM NaCl for 5 h at 37°C prior to electrophoretic analysis.
    Figure Legend Snippet: ( A ) Agarose gel electrophoresis of supercoiled (400 ng) and ( B ) a 742 bp dsDNA fragment of pUC19 (400 ng) exposed to increasing concentrations of MC3, OC3 and HC3 . Reactions were carried out in the presence of 25 mM NaCl for 5 h at 37°C prior to electrophoretic analysis.

    Techniques Used: Agarose Gel Electrophoresis

    Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .
    Figure Legend Snippet: Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .

    Techniques Used: Microscopy

    8) Product Images from "Impaired STING Pathway in Human Osteosarcoma U2OS Cells Contributes to the Growth of ICP0-Null Mutant Herpes Simplex Virus"

    Article Title: Impaired STING Pathway in Human Osteosarcoma U2OS Cells Contributes to the Growth of ICP0-Null Mutant Herpes Simplex Virus

    Journal: Journal of Virology

    doi: 10.1128/JVI.00006-17

    Restoration of STING expression in U2OS cells rescues innate immunity. (A) The U2OS cells were either mock transfected (lanes 2 to 4) or transfected with STING (lanes 8 to 10), IFI16-expressing plasmids (lanes 11 to 13), or pUC19 (lanes 5 to 7) as a control.
    Figure Legend Snippet: Restoration of STING expression in U2OS cells rescues innate immunity. (A) The U2OS cells were either mock transfected (lanes 2 to 4) or transfected with STING (lanes 8 to 10), IFI16-expressing plasmids (lanes 11 to 13), or pUC19 (lanes 5 to 7) as a control.

    Techniques Used: Expressing, Transfection

    9) Product Images from "Role for FimH in Extraintestinal Pathogenic Escherichia coli Invasion and Translocation through the Intestinal Epithelium"

    Article Title: Role for FimH in Extraintestinal Pathogenic Escherichia coli Invasion and Translocation through the Intestinal Epithelium

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00581-17

    FimH mediates ExPEC invasion of human jejunal intestinal enteroid monolayers (HJIEMs). HJIEMs were infected at an MOI of 100 with ExPEC wild-type (Wt) or Δ fimH strain or pUC19:Δ fimH ExPEC strain CP9, and adherence (A) and invasion (B) were measured (see Materials and Methods). (C) The effects of mannose on wild-type (Wt) ExPEC and Δ fimH mutant strains were evaluated as described in Materials and Methods. Data are means ± SEM; n = 4 to 8, assayed in triplicate; *, P ≤ 0.05.
    Figure Legend Snippet: FimH mediates ExPEC invasion of human jejunal intestinal enteroid monolayers (HJIEMs). HJIEMs were infected at an MOI of 100 with ExPEC wild-type (Wt) or Δ fimH strain or pUC19:Δ fimH ExPEC strain CP9, and adherence (A) and invasion (B) were measured (see Materials and Methods). (C) The effects of mannose on wild-type (Wt) ExPEC and Δ fimH mutant strains were evaluated as described in Materials and Methods. Data are means ± SEM; n = 4 to 8, assayed in triplicate; *, P ≤ 0.05.

    Techniques Used: Infection, Mutagenesis

    FimH is important for ExPEC translocation of an immunocompromised host. (A to G) BALB/c mice were subjected to oral gavage with wild-type (Wt), fimH mutant, or complemented pUC19:Δ fimH ExPEC strains at 10 9 CFU/100 μl. Mice were given cyclophosphamide at a total dose of 450 mg/kg (three 150-mg/kg doses on days 1, 3, and 5) by intraperitoneal injection. Intestinal colonization was measured by fecal homogenates (days 1, 3, 5, and 7) (A) or intestinal homogenates (8 days postinfection immediately after euthanasia) (B, C) plated on LB/agar plus 10 μg/ml CM. (D to G) Translocation out of the GI tract was determined by the presence of bacteria in organs as described in Materials and Methods. Data are means ± SEM; n = 14 to 16 for each group. *, P ≤ 0.05; **, P ≤ 0.01. (H) For an intraperitoneal infection, BALB/c mice were given 10 7 CFU/100 μl of wild-type (Wt) ExPEC or fimH mutant strains. Colonization was measured by organ homogenates plated on LB/agar plus 10 μg/ml CM (see Materials and Methods). Data are means ± SEM; n = 8 for each group.
    Figure Legend Snippet: FimH is important for ExPEC translocation of an immunocompromised host. (A to G) BALB/c mice were subjected to oral gavage with wild-type (Wt), fimH mutant, or complemented pUC19:Δ fimH ExPEC strains at 10 9 CFU/100 μl. Mice were given cyclophosphamide at a total dose of 450 mg/kg (three 150-mg/kg doses on days 1, 3, and 5) by intraperitoneal injection. Intestinal colonization was measured by fecal homogenates (days 1, 3, 5, and 7) (A) or intestinal homogenates (8 days postinfection immediately after euthanasia) (B, C) plated on LB/agar plus 10 μg/ml CM. (D to G) Translocation out of the GI tract was determined by the presence of bacteria in organs as described in Materials and Methods. Data are means ± SEM; n = 14 to 16 for each group. *, P ≤ 0.05; **, P ≤ 0.01. (H) For an intraperitoneal infection, BALB/c mice were given 10 7 CFU/100 μl of wild-type (Wt) ExPEC or fimH mutant strains. Colonization was measured by organ homogenates plated on LB/agar plus 10 μg/ml CM (see Materials and Methods). Data are means ± SEM; n = 8 for each group.

    Techniques Used: Translocation Assay, Mouse Assay, Mutagenesis, Injection, Infection

    10) Product Images from "C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents"

    Article Title: C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1097

    ( A ) Influence of ionic strength on pUC19 condensation (400 ng) by OC3 and MC3 (25 μM) opioid compounds. Condensation reactions on pUC19 (400 ng) by opioid compounds in ( B ) acidic NaOAc buffer (80 mM, pH = 4.0), and ( C ) basic Tris buffer (80 mM, pH = 9.0) in the presence of 25 mM NaCl.
    Figure Legend Snippet: ( A ) Influence of ionic strength on pUC19 condensation (400 ng) by OC3 and MC3 (25 μM) opioid compounds. Condensation reactions on pUC19 (400 ng) by opioid compounds in ( B ) acidic NaOAc buffer (80 mM, pH = 4.0), and ( C ) basic Tris buffer (80 mM, pH = 9.0) in the presence of 25 mM NaCl.

    Techniques Used:

    ( A ) Agarose gel electrophoresis of supercoiled (400 ng) and ( B ) a 742 bp dsDNA fragment of pUC19 (400 ng) exposed to increasing concentrations of MC3, OC3 and HC3 . Reactions were carried out in the presence of 25 mM NaCl for 5 h at 37°C prior to electrophoretic analysis.
    Figure Legend Snippet: ( A ) Agarose gel electrophoresis of supercoiled (400 ng) and ( B ) a 742 bp dsDNA fragment of pUC19 (400 ng) exposed to increasing concentrations of MC3, OC3 and HC3 . Reactions were carried out in the presence of 25 mM NaCl for 5 h at 37°C prior to electrophoretic analysis.

    Techniques Used: Agarose Gel Electrophoresis

    Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .
    Figure Legend Snippet: Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .

    Techniques Used: Microscopy

    11) Product Images from "C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents"

    Article Title: C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1097

    ( A ) Influence of ionic strength on pUC19 condensation (400 ng) by OC3 and MC3 (25 μM) opioid compounds. Condensation reactions on pUC19 (400 ng) by opioid compounds in ( B ) acidic NaOAc buffer (80 mM, pH = 4.0), and ( C ) basic Tris buffer (80 mM, pH = 9.0) in the presence of 25 mM NaCl.
    Figure Legend Snippet: ( A ) Influence of ionic strength on pUC19 condensation (400 ng) by OC3 and MC3 (25 μM) opioid compounds. Condensation reactions on pUC19 (400 ng) by opioid compounds in ( B ) acidic NaOAc buffer (80 mM, pH = 4.0), and ( C ) basic Tris buffer (80 mM, pH = 9.0) in the presence of 25 mM NaCl.

    Techniques Used:

    ( A ) Agarose gel electrophoresis of supercoiled (400 ng) and ( B ) a 742 bp dsDNA fragment of pUC19 (400 ng) exposed to increasing concentrations of MC3, OC3 and HC3 . Reactions were carried out in the presence of 25 mM NaCl for 5 h at 37°C prior to electrophoretic analysis.
    Figure Legend Snippet: ( A ) Agarose gel electrophoresis of supercoiled (400 ng) and ( B ) a 742 bp dsDNA fragment of pUC19 (400 ng) exposed to increasing concentrations of MC3, OC3 and HC3 . Reactions were carried out in the presence of 25 mM NaCl for 5 h at 37°C prior to electrophoretic analysis.

    Techniques Used: Agarose Gel Electrophoresis

    Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .
    Figure Legend Snippet: Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .

    Techniques Used: Microscopy

    12) Product Images from "Ciprofloxacin is an inhibitor of the Mcm2-7 replicative helicase"

    Article Title: Ciprofloxacin is an inhibitor of the Mcm2-7 replicative helicase

    Journal: Bioscience Reports

    doi: 10.1042/BSR20130083

    Mode of action of the various small molecule inhibitors Treatment order for each panel: 0, solvent control; 1, compound 125248; 2, 924384; 3, MAL2-11b; 4, 268973; 5, 388612; 6, 314850; 7, 271327; and 8, ciprofloxacin. ( A ) Effects of each inhibitor on steady-state ATP turnover by Mcm2-7, Mcm467 and TAg. This experiment was identical to that shown in Figure 1 (C) with the indicated helicase complexes used at 100 nM concentration, but 1 mM of the indicated inhibitor was added prior to ATP addition. Bar graphs show the levels of ATP hydrolysis observed after 30 min of incubation as a % of the ATP hydrolysis observed in the absence of inhibitor (treatment 0). ( B ) Effect of increased ATP concentration with indicated inhibitor on DNA unwinding activity of Mcm2-7. The standard helicase reaction was supplemented with the indicated inhibitor concentration (numbered 1–8 as in A) in the presence (+) or absence of an additional 5 mM ATP. ATP and the indicated inhibitor were added together to Mcm2-7 without preincubation. ‘Fold’ refers to the ratio of DNA unwinding between the reactions containing 10 mM ATP and containing 5 mM ATP. ( C ) Ability of inhibitors to intercalate into DNA. In the intercalation assay (Experimental Procedures), Topo I (4 units) was used to relax 50 ng of monomeric pUC19 (treatment 0; compare supercoiled DNA (left) with relaxed DNA (right)). After 1 h of Topo I treatment, 1mM of the indicated inhibitor was added and samples were incubated for an additional 1 h (D) Topo I activity inhibition assay. This experiment was identical to ( C ), except that Topo I and the indicated inhibitor were added at the same time. Topo I inhibition is indicated if addition of both inhibitor and topoisomerase together generates supercoiled DNA, while experiments shown in ( C ) generate relaxed plasmid. ( E ) An intercalation assay performed with the indicated inhibitors at lower concentrations. These assays were similar to ( C ) (Topo I added first, and the inhibitor added second), except the indicated concentration of inhibitor was used.
    Figure Legend Snippet: Mode of action of the various small molecule inhibitors Treatment order for each panel: 0, solvent control; 1, compound 125248; 2, 924384; 3, MAL2-11b; 4, 268973; 5, 388612; 6, 314850; 7, 271327; and 8, ciprofloxacin. ( A ) Effects of each inhibitor on steady-state ATP turnover by Mcm2-7, Mcm467 and TAg. This experiment was identical to that shown in Figure 1 (C) with the indicated helicase complexes used at 100 nM concentration, but 1 mM of the indicated inhibitor was added prior to ATP addition. Bar graphs show the levels of ATP hydrolysis observed after 30 min of incubation as a % of the ATP hydrolysis observed in the absence of inhibitor (treatment 0). ( B ) Effect of increased ATP concentration with indicated inhibitor on DNA unwinding activity of Mcm2-7. The standard helicase reaction was supplemented with the indicated inhibitor concentration (numbered 1–8 as in A) in the presence (+) or absence of an additional 5 mM ATP. ATP and the indicated inhibitor were added together to Mcm2-7 without preincubation. ‘Fold’ refers to the ratio of DNA unwinding between the reactions containing 10 mM ATP and containing 5 mM ATP. ( C ) Ability of inhibitors to intercalate into DNA. In the intercalation assay (Experimental Procedures), Topo I (4 units) was used to relax 50 ng of monomeric pUC19 (treatment 0; compare supercoiled DNA (left) with relaxed DNA (right)). After 1 h of Topo I treatment, 1mM of the indicated inhibitor was added and samples were incubated for an additional 1 h (D) Topo I activity inhibition assay. This experiment was identical to ( C ), except that Topo I and the indicated inhibitor were added at the same time. Topo I inhibition is indicated if addition of both inhibitor and topoisomerase together generates supercoiled DNA, while experiments shown in ( C ) generate relaxed plasmid. ( E ) An intercalation assay performed with the indicated inhibitors at lower concentrations. These assays were similar to ( C ) (Topo I added first, and the inhibitor added second), except the indicated concentration of inhibitor was used.

    Techniques Used: Concentration Assay, Incubation, Activity Assay, Inhibition, Plasmid Preparation

    13) Product Images from "Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori"

    Article Title: Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw730

    ( A ) Binding of TieA to dsDNA: electrophoretic mobility shift assays were carried out by incubating different concentrations of TieA (0.1, 0.5, 1 and 2 μg) with 0.5 nM 32 P-labeled DNA substrates. Samples were subjected to electrophoresis on native PAGE and visualized by autoradiography as mentioned in materials and methods section. ( B ) TieA binds to DNA non-specifically: electrophoretic mobility shift assays were carried out by incubating 1 μg of TieA with mutated oligos 1–5 (see Supplementary Table S1). ( C ) Nuclease activity of TieA: different concentrations of TieA (0.01, 0.1, 0.2, 0.5, 1 and 2 μg corresponding to lanes 7-12, respectively) were incubated with 1 μg of pUC19 DNA for 1 h at 37 °C. The reaction was stopped by addition of 10 mM EDTA and samples were deprotonized by adding proteinase K (10 μg/sample) in presence of 0.05% SDS for 15 min at 65°C. The digested products were separated on 1.2% agarose gel. Rv3131 (0.5 μg) was used as a negative control in lane 6. MboII (1 unit/reaction) and DNase I (1 unit/reaction) served as positive controls in lanes 3 and 5, respectively. Lane 4 represents heat inactivated TieA. ( D ) TieA cleaves both pUC19 (circular) and Lambda DNA (linear): pUC19 and Lambda DNA were incubated with TieA (lanes 5, 6, 14 and 15) for 1 h at 37°C and processed as described above. MboII (lanes 3 and 12) and DNase I (lanes 4 and 13) were used as positive controls. Rv3131 protein was used as a negative control (lanes 7 and 16). Ca 2+ –Mg 2+ dependent nuclease activity of TieA was confirmed by pre-incubating pUC19/Lambda DNA with either SDS (0.05%) or EDTA (10 mM) for 10 min (lanes 8, 9, 17 and 18) and later 1 μg of TieA was added and further processed as described above. Data are representative of three independent experiments. HI: heat inactivated.
    Figure Legend Snippet: ( A ) Binding of TieA to dsDNA: electrophoretic mobility shift assays were carried out by incubating different concentrations of TieA (0.1, 0.5, 1 and 2 μg) with 0.5 nM 32 P-labeled DNA substrates. Samples were subjected to electrophoresis on native PAGE and visualized by autoradiography as mentioned in materials and methods section. ( B ) TieA binds to DNA non-specifically: electrophoretic mobility shift assays were carried out by incubating 1 μg of TieA with mutated oligos 1–5 (see Supplementary Table S1). ( C ) Nuclease activity of TieA: different concentrations of TieA (0.01, 0.1, 0.2, 0.5, 1 and 2 μg corresponding to lanes 7-12, respectively) were incubated with 1 μg of pUC19 DNA for 1 h at 37 °C. The reaction was stopped by addition of 10 mM EDTA and samples were deprotonized by adding proteinase K (10 μg/sample) in presence of 0.05% SDS for 15 min at 65°C. The digested products were separated on 1.2% agarose gel. Rv3131 (0.5 μg) was used as a negative control in lane 6. MboII (1 unit/reaction) and DNase I (1 unit/reaction) served as positive controls in lanes 3 and 5, respectively. Lane 4 represents heat inactivated TieA. ( D ) TieA cleaves both pUC19 (circular) and Lambda DNA (linear): pUC19 and Lambda DNA were incubated with TieA (lanes 5, 6, 14 and 15) for 1 h at 37°C and processed as described above. MboII (lanes 3 and 12) and DNase I (lanes 4 and 13) were used as positive controls. Rv3131 protein was used as a negative control (lanes 7 and 16). Ca 2+ –Mg 2+ dependent nuclease activity of TieA was confirmed by pre-incubating pUC19/Lambda DNA with either SDS (0.05%) or EDTA (10 mM) for 10 min (lanes 8, 9, 17 and 18) and later 1 μg of TieA was added and further processed as described above. Data are representative of three independent experiments. HI: heat inactivated.

    Techniques Used: Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Electrophoresis, Clear Native PAGE, Autoradiography, Activity Assay, Incubation, Agarose Gel Electrophoresis, Negative Control, Lambda DNA Preparation

    14) Product Images from "DNA looping by Ku and the DNA-dependent protein kinase"

    Article Title: DNA looping by Ku and the DNA-dependent protein kinase

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    AFM and electron microscopy of DNA-PK complexes. ( A ) Reactions containing DNA-PKcs and a linear 660-bp DNA fragment were examined by AFM. Association between DNA-PKcs and DNA (arrow) was observed but uncommon compared with reactions that also contained Ku. ( B ) In the presence of recombinant Ku, DNA-PKcs was frequently seen associated with the DNA fragment (white arrows). Black arrows indicate unbound DNA-PKcs. Samples in A and B were mounted and imaged without fixation. ( C ) Linearized pUC19 incubated in the presence of Ku and DNA-PKcs. Size differences allowed the DNA-PKcs (black arrowhead) to be distinguished from recombinant Ku particles (white arrowhead). End-bound (long arrow) and internally bound DNA-PK (short arrow) were both commonly observed. ( D and E ) Electron microscopy of rotary-shadowed DNA-PK/DNA complexes assembled using HeLa-purified Ku revealed similar DNA-PK behavior. ( D ) A DNA-PK complex bound internally to linearized pUC19. The arrow indicates the DNA end. ( E ) An example of an end-bound DNA-PK complex. Bar in A is 100 nm for A – C . Bar in D is 100 nm for D and E .
    Figure Legend Snippet: AFM and electron microscopy of DNA-PK complexes. ( A ) Reactions containing DNA-PKcs and a linear 660-bp DNA fragment were examined by AFM. Association between DNA-PKcs and DNA (arrow) was observed but uncommon compared with reactions that also contained Ku. ( B ) In the presence of recombinant Ku, DNA-PKcs was frequently seen associated with the DNA fragment (white arrows). Black arrows indicate unbound DNA-PKcs. Samples in A and B were mounted and imaged without fixation. ( C ) Linearized pUC19 incubated in the presence of Ku and DNA-PKcs. Size differences allowed the DNA-PKcs (black arrowhead) to be distinguished from recombinant Ku particles (white arrowhead). End-bound (long arrow) and internally bound DNA-PK (short arrow) were both commonly observed. ( D and E ) Electron microscopy of rotary-shadowed DNA-PK/DNA complexes assembled using HeLa-purified Ku revealed similar DNA-PK behavior. ( D ) A DNA-PK complex bound internally to linearized pUC19. The arrow indicates the DNA end. ( E ) An example of an end-bound DNA-PK complex. Bar in A is 100 nm for A – C . Bar in D is 100 nm for D and E .

    Techniques Used: Electron Microscopy, Recombinant, Incubation, Purification

    15) Product Images from "Ehrlichia chaffeensis Proliferation Begins with NtrY/NtrX and PutA/GlnA Upregulation and CtrA Degradation Induced by Proline and Glutamine Uptake"

    Article Title: Ehrlichia chaffeensis Proliferation Begins with NtrY/NtrX and PutA/GlnA Upregulation and CtrA Degradation Induced by Proline and Glutamine Uptake

    Journal: mBio

    doi: 10.1128/mBio.02141-14

    E. chaffeensis PutA and GlnA are functional enzymes, and E. chaffeensis GlnA is sensitive to MSX. (A) E. chaffeensis PutA complements an E. coli putA mutant (JT31). wt (CSH4) and JT31 transformed with the E. chaffeensis putA construct or pUC19 (negative control) were cultured on redox indicator TTC-proline plates. (B) E. chaffeensis GlnA complements an E. coli glnA mutant (YMC11). wt (XL1-Blue) and YMC11 transformed with the E. chaffeensis glnA construct or pUC19 (negative control) were cultured on M9 plates containing 20 mM (NH4) 2 SO 4 as the sole nitrogen source. (C) MSX inhibits E. chaffeensis GlnA activity. YMC11 transformed with the E. chaffeensis glnA construct was cultured on M9 plates containing 2 mM glutamine (Gln) or 20 mM (NH4) 2 SO 4 with or without 200 µM MSX.
    Figure Legend Snippet: E. chaffeensis PutA and GlnA are functional enzymes, and E. chaffeensis GlnA is sensitive to MSX. (A) E. chaffeensis PutA complements an E. coli putA mutant (JT31). wt (CSH4) and JT31 transformed with the E. chaffeensis putA construct or pUC19 (negative control) were cultured on redox indicator TTC-proline plates. (B) E. chaffeensis GlnA complements an E. coli glnA mutant (YMC11). wt (XL1-Blue) and YMC11 transformed with the E. chaffeensis glnA construct or pUC19 (negative control) were cultured on M9 plates containing 20 mM (NH4) 2 SO 4 as the sole nitrogen source. (C) MSX inhibits E. chaffeensis GlnA activity. YMC11 transformed with the E. chaffeensis glnA construct was cultured on M9 plates containing 2 mM glutamine (Gln) or 20 mM (NH4) 2 SO 4 with or without 200 µM MSX.

    Techniques Used: Functional Assay, Mutagenesis, Transformation Assay, Construct, Negative Control, Cell Culture, Activity Assay

    16) Product Images from "C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents"

    Article Title: C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1097

    ( A ) Influence of ionic strength on pUC19 condensation (400 ng) by OC3 and MC3 (25 μM) opioid compounds. Condensation reactions on pUC19 (400 ng) by opioid compounds in ( B ) acidic NaOAc buffer (80 mM, pH = 4.0), and ( C ) basic Tris buffer (80 mM, pH = 9.0) in the presence of 25 mM NaCl.
    Figure Legend Snippet: ( A ) Influence of ionic strength on pUC19 condensation (400 ng) by OC3 and MC3 (25 μM) opioid compounds. Condensation reactions on pUC19 (400 ng) by opioid compounds in ( B ) acidic NaOAc buffer (80 mM, pH = 4.0), and ( C ) basic Tris buffer (80 mM, pH = 9.0) in the presence of 25 mM NaCl.

    Techniques Used:

    ( A ) Agarose gel electrophoresis of supercoiled (400 ng) and ( B ) a 742 bp dsDNA fragment of pUC19 (400 ng) exposed to increasing concentrations of MC3, OC3 and HC3 . Reactions were carried out in the presence of 25 mM NaCl for 5 h at 37°C prior to electrophoretic analysis.
    Figure Legend Snippet: ( A ) Agarose gel electrophoresis of supercoiled (400 ng) and ( B ) a 742 bp dsDNA fragment of pUC19 (400 ng) exposed to increasing concentrations of MC3, OC3 and HC3 . Reactions were carried out in the presence of 25 mM NaCl for 5 h at 37°C prior to electrophoretic analysis.

    Techniques Used: Agarose Gel Electrophoresis

    Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .
    Figure Legend Snippet: Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .

    Techniques Used: Microscopy

    17) Product Images from "Selective Microbial Genomic DNA Isolation Using Restriction Endonucleases"

    Article Title: Selective Microbial Genomic DNA Isolation Using Restriction Endonucleases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0109061

    Analysis of biotinylated DpnI. (A) pUC19 was incubated with DpnI, DpnI-biotin or commercially sourced DpnI in the presence or absence of 10 mM magnesium chloride. The digested fragments were separated on a 1.5% agarose gel. (B) A FAM-labeled DNA duplex containing one G m6 ATC site was incubated with increasing amounts of DpnI or DpnI-biotin (0 to 1200 ng). The reactions were separated on a 20% TBE gel and analyzed with fluorescence imaging. (C) An unmethylated 651 bp DNA fragment and a Dam-methylated 477 bp DNA fragment were combined and incubated with increasing amounts of immobilized DpnI-biotin (80–180 µl). DNA was eluted using GTC and desalted. All fractions were separated on a 3% agarose gel.
    Figure Legend Snippet: Analysis of biotinylated DpnI. (A) pUC19 was incubated with DpnI, DpnI-biotin or commercially sourced DpnI in the presence or absence of 10 mM magnesium chloride. The digested fragments were separated on a 1.5% agarose gel. (B) A FAM-labeled DNA duplex containing one G m6 ATC site was incubated with increasing amounts of DpnI or DpnI-biotin (0 to 1200 ng). The reactions were separated on a 20% TBE gel and analyzed with fluorescence imaging. (C) An unmethylated 651 bp DNA fragment and a Dam-methylated 477 bp DNA fragment were combined and incubated with increasing amounts of immobilized DpnI-biotin (80–180 µl). DNA was eluted using GTC and desalted. All fractions were separated on a 3% agarose gel.

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Labeling, Fluorescence, Imaging, Methylation

    18) Product Images from "CRISPR-Cas12a Possesses Unconventional DNase Activity that Can Be Inactivated by Synthetic Oligonucleotides"

    Article Title: CRISPR-Cas12a Possesses Unconventional DNase Activity that Can Be Inactivated by Synthetic Oligonucleotides

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2019.12.038

    Time Course of Degradation of DNA Substrates by Cas12a (A and B) Representative gel images of Cas12a activity toward M13mp18 ssDNA (A) and pUC19 dsDNA (B) at indicated time points (min) were shown in the upper panel. Positions of M13, supercoiled (SC), nicked (N), and linear (L) DNA were indicated. The vertical dotted line indicates the border between two separate gels. The fraction cleaved (%) from three independent experiments is plotted against the time points (min) and fitted with a nonlinear regression in the lower panel. Error bars are presented as mean ± SEM.
    Figure Legend Snippet: Time Course of Degradation of DNA Substrates by Cas12a (A and B) Representative gel images of Cas12a activity toward M13mp18 ssDNA (A) and pUC19 dsDNA (B) at indicated time points (min) were shown in the upper panel. Positions of M13, supercoiled (SC), nicked (N), and linear (L) DNA were indicated. The vertical dotted line indicates the border between two separate gels. The fraction cleaved (%) from three independent experiments is plotted against the time points (min) and fitted with a nonlinear regression in the lower panel. Error bars are presented as mean ± SEM.

    Techniques Used: Activity Assay

    Inhibitory Effects of Anti-Cas12a psDNA on Cas12a-Mediated DNase Activity toward Linear DNA (A–C) The effects of anti-Cas12a psDNA on metal-dependent ssDNase activity of Cas12a orthologs (AsCas12a, LbCas12a, and FnCas12a) toward linear ssDNA (A), dsDNA (B), and linearized pUC19 (C).
    Figure Legend Snippet: Inhibitory Effects of Anti-Cas12a psDNA on Cas12a-Mediated DNase Activity toward Linear DNA (A–C) The effects of anti-Cas12a psDNA on metal-dependent ssDNase activity of Cas12a orthologs (AsCas12a, LbCas12a, and FnCas12a) toward linear ssDNA (A), dsDNA (B), and linearized pUC19 (C).

    Techniques Used: Activity Assay

    crRNA-Free DNase Activities of Cas12a Orthologs toward Circular DNA (A) Representative in vitro cleavage of circular phage M13mp18 ssDNA by AsCas12a and LbCas12a in the presence of various divalent metal ions. FnCas12a was used as a positive group. Control (Ctrl), M13mp18 ssDNA alone. (B) Metal ion concentration dependence for cleavage of M13mp18 ssDNA substrates by AsCas12a and LbCas12a. Different concentrations of Mg 2+ or Mn 2+ ranging from 0.5 to 10 mM were used. Ctrl, M13mp18 ssDNA alone. (C) Representative in vitro cleavage of circular plasmid pUC19 dsDNA by AsCas12a and LbCas12a in the presence of various divalent metal ions. FnCas12a was used as a positive group. Ctrl, pUC19 dsDNA alone. (D) Metal ion concentration dependence for cleavage of pUC19 dsDNA substrates by AsCas12a and LbCas12a. Different concentrations of Mg 2+ or Mn 2+ ranging from 0.5 to 10 mM were used. Ctrl, pUC19 dsDNA alone. Positions of M13mp18, supercoiled (SC, green arrow), nicked (N, red arrow), and linear (L, red arrow) DNA were indicated.
    Figure Legend Snippet: crRNA-Free DNase Activities of Cas12a Orthologs toward Circular DNA (A) Representative in vitro cleavage of circular phage M13mp18 ssDNA by AsCas12a and LbCas12a in the presence of various divalent metal ions. FnCas12a was used as a positive group. Control (Ctrl), M13mp18 ssDNA alone. (B) Metal ion concentration dependence for cleavage of M13mp18 ssDNA substrates by AsCas12a and LbCas12a. Different concentrations of Mg 2+ or Mn 2+ ranging from 0.5 to 10 mM were used. Ctrl, M13mp18 ssDNA alone. (C) Representative in vitro cleavage of circular plasmid pUC19 dsDNA by AsCas12a and LbCas12a in the presence of various divalent metal ions. FnCas12a was used as a positive group. Ctrl, pUC19 dsDNA alone. (D) Metal ion concentration dependence for cleavage of pUC19 dsDNA substrates by AsCas12a and LbCas12a. Different concentrations of Mg 2+ or Mn 2+ ranging from 0.5 to 10 mM were used. Ctrl, pUC19 dsDNA alone. Positions of M13mp18, supercoiled (SC, green arrow), nicked (N, red arrow), and linear (L, red arrow) DNA were indicated.

    Techniques Used: In Vitro, Concentration Assay, Plasmid Preparation

    Inhibitory Effects of Anti-Cas12a psDNA on Cas12a-Mediated DNase Activity toward Circular DNA (A) Dose-dependent inhibitory effects of anti-Cas12a psDNA on Mg 2+ -promoted AsCas12a activity. Control (Ctrl), M13mp18 ssDNA alone. ssDNA1 was parallelly used to rule out the possibility of oligonucleotides interference. The concentration of AsCas12a was 200 nM. (B–D) The effects of anti-Cas12a psDNA on metal-dependent ssDNase activity of Cas12a orthologs (AsCas12a, LbCas12a, and FnCas12a) on circular ssDNA M13mp18 (B), ΦX174 (C), and dsDNA pUC19 (D). The concentration of anti-Cas12a psDNA was 200 nM. Ctrl, M13mp18 ssDNA alone. The vertical dotted line indicates the border between two separate gels. Ctrl, pUC19 dsDNA alone. Positions of M13, ΦX174, supercoiled (SC), nicked (N), and linear (L) DNA were indicated.
    Figure Legend Snippet: Inhibitory Effects of Anti-Cas12a psDNA on Cas12a-Mediated DNase Activity toward Circular DNA (A) Dose-dependent inhibitory effects of anti-Cas12a psDNA on Mg 2+ -promoted AsCas12a activity. Control (Ctrl), M13mp18 ssDNA alone. ssDNA1 was parallelly used to rule out the possibility of oligonucleotides interference. The concentration of AsCas12a was 200 nM. (B–D) The effects of anti-Cas12a psDNA on metal-dependent ssDNase activity of Cas12a orthologs (AsCas12a, LbCas12a, and FnCas12a) on circular ssDNA M13mp18 (B), ΦX174 (C), and dsDNA pUC19 (D). The concentration of anti-Cas12a psDNA was 200 nM. Ctrl, M13mp18 ssDNA alone. The vertical dotted line indicates the border between two separate gels. Ctrl, pUC19 dsDNA alone. Positions of M13, ΦX174, supercoiled (SC), nicked (N), and linear (L) DNA were indicated.

    Techniques Used: Activity Assay, Concentration Assay

    19) Product Images from "Investigating the endonuclease activity of four Pyrococcus abyssi inteins"

    Article Title: Investigating the endonuclease activity of four Pyrococcus abyssi inteins

    Journal: Nucleic Acids Research

    doi:

    Standard cleavage assays for PI- Pab I ( Pab RIR1-2) and PI- Pab II ( Pab RIR1-3). Cleavage assays for PI- Pab I on its circular substrate ( A ), linear substrate ( B ) and on linear plasmid pUC19 ( C ). 100 ng of purified pSiteC plasmid was incubated with 0.10 ng (A), 0.40 ng (B) or 15 ng (C) of PI- Pab I for different times at 70°C, in a 5 mM Tris–acetate pH 9.5 buffer containing 5 mM KCl and 5 mM MgCl 2 . Cleavage assays for PI- Pab II on its circular substrate ( D ), linear substrate ( E ) and on linear plasmid pUC19 ( F ). 100 ng of purified pSiteD plasmid were incubated with 1 ng [(D) and (E)] or 15 ng (F) of PI- Pab II for different times at 70°C, in a 10 mM Tris–HCl pH 8 buffer containing 25 mM NH 4 OAc and 2 mM MgCl 2 . Either supercoiled (sc), open circular (oc) and linear (lin) forms of DNA or linear substrate (S, 2710 bp) and fragment products (P, 960 and 1750 bp) were separated on a 1% agarose gel in 0.5× TBE buffer.
    Figure Legend Snippet: Standard cleavage assays for PI- Pab I ( Pab RIR1-2) and PI- Pab II ( Pab RIR1-3). Cleavage assays for PI- Pab I on its circular substrate ( A ), linear substrate ( B ) and on linear plasmid pUC19 ( C ). 100 ng of purified pSiteC plasmid was incubated with 0.10 ng (A), 0.40 ng (B) or 15 ng (C) of PI- Pab I for different times at 70°C, in a 5 mM Tris–acetate pH 9.5 buffer containing 5 mM KCl and 5 mM MgCl 2 . Cleavage assays for PI- Pab II on its circular substrate ( D ), linear substrate ( E ) and on linear plasmid pUC19 ( F ). 100 ng of purified pSiteD plasmid were incubated with 1 ng [(D) and (E)] or 15 ng (F) of PI- Pab II for different times at 70°C, in a 10 mM Tris–HCl pH 8 buffer containing 25 mM NH 4 OAc and 2 mM MgCl 2 . Either supercoiled (sc), open circular (oc) and linear (lin) forms of DNA or linear substrate (S, 2710 bp) and fragment products (P, 960 and 1750 bp) were separated on a 1% agarose gel in 0.5× TBE buffer.

    Techniques Used: Plasmid Preparation, Purification, Incubation, Agarose Gel Electrophoresis

    20) Product Images from "EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA"

    Article Title: EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA

    Journal: bioRxiv

    doi: 10.1101/2019.12.20.884692

    EM-seq accurately represents methylation EM-seq and bisulfite libraries were made using 10, 50 and 200 ng of NA12878 DNA with control DNA (2 ng unmethylated lambda and 0.1 ng CpG methylated pUC19). Libraries were sequenced on an Illumina NovaSeq 6000 (2 x 100 bases). 324 million paired reads for each library were aligned to a human + control reference genome (see supplemental materials) using bwa-meth 0.2.2. and methylation information was extracted from the alignments using MethylDackel. The top and bottom strand CpGs were counted independently, yielding a maximum of 56 million possible CpG sites. (A) NA12878 EM-seq and whole genome bisulfite library (WGBS) methylation in CpG, CHH and CHG contexts are similarly represented. Methylation state for unmethylated lambda control and CpG methylated pUC19 control DNAs are shown in Supplemental Figure 5. (B) The number of CpGs covered for EM-seq and bisulfite libraries were calculated and graphed at minimum coverage depths of 1x through 21x. (C) The number of CpGs detected were compared between EM-seq and bisulfite libraries at 1x and 8x coverage depths. CpGs unique to EM-seq libraries, bisulfite libraries or those that were common to both are represented in the Venn diagrams. (D, E) Methylkit analysis at minimum 1x coverage shows good CpG methylation correlation between 10 ng and 200 ng NA12878 EM-seq libraries (D) and WGBS libraries (E). Methylation level correlations between inputs and replicates of EM-seq libraries are better than for WGBS libraries. The reduction in observations of disagreement (upper left and lower right corners) is particularly striking. Correlation between EM-seq and WGBS libraries at 10 ng, 50 ng, and 200 ng NA12878 DNA input are shown in Supplemental Figure 7.
    Figure Legend Snippet: EM-seq accurately represents methylation EM-seq and bisulfite libraries were made using 10, 50 and 200 ng of NA12878 DNA with control DNA (2 ng unmethylated lambda and 0.1 ng CpG methylated pUC19). Libraries were sequenced on an Illumina NovaSeq 6000 (2 x 100 bases). 324 million paired reads for each library were aligned to a human + control reference genome (see supplemental materials) using bwa-meth 0.2.2. and methylation information was extracted from the alignments using MethylDackel. The top and bottom strand CpGs were counted independently, yielding a maximum of 56 million possible CpG sites. (A) NA12878 EM-seq and whole genome bisulfite library (WGBS) methylation in CpG, CHH and CHG contexts are similarly represented. Methylation state for unmethylated lambda control and CpG methylated pUC19 control DNAs are shown in Supplemental Figure 5. (B) The number of CpGs covered for EM-seq and bisulfite libraries were calculated and graphed at minimum coverage depths of 1x through 21x. (C) The number of CpGs detected were compared between EM-seq and bisulfite libraries at 1x and 8x coverage depths. CpGs unique to EM-seq libraries, bisulfite libraries or those that were common to both are represented in the Venn diagrams. (D, E) Methylkit analysis at minimum 1x coverage shows good CpG methylation correlation between 10 ng and 200 ng NA12878 EM-seq libraries (D) and WGBS libraries (E). Methylation level correlations between inputs and replicates of EM-seq libraries are better than for WGBS libraries. The reduction in observations of disagreement (upper left and lower right corners) is particularly striking. Correlation between EM-seq and WGBS libraries at 10 ng, 50 ng, and 200 ng NA12878 DNA input are shown in Supplemental Figure 7.

    Techniques Used: Methylation, CpG Methylation Assay

    21) Product Images from "Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes"

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-19920-y

    Synthetic c ysE and cysM gene transformants display recovery of CysE function without cysteine and methionine and CysM function without cysteine. ( A ) E. coli ΔcysE competent cells were transformed with positive control cysE , two cysE variants cysE-C/cysE-CM cloned into the multiple cloning site of pUC19 plasmid, and original pUC19 encoding N-terminal fragment of lacZ α as a negative control. ( B ) E. coli ΔcysMΔcysK competent cells transformed with positive control c ysM , two cysM variants cysM-C / cysM-CM in pUC19 plasmid, and original pUC19 encoding N-terminal fragment of lacZ α as a negative control. Cells were plated on M9 + glucose medium with 0.4 mM IPTG, 50 μg/ml kanamycin, and 100 μg/ml ampicillin and incubated at 30 °C for 72 h.
    Figure Legend Snippet: Synthetic c ysE and cysM gene transformants display recovery of CysE function without cysteine and methionine and CysM function without cysteine. ( A ) E. coli ΔcysE competent cells were transformed with positive control cysE , two cysE variants cysE-C/cysE-CM cloned into the multiple cloning site of pUC19 plasmid, and original pUC19 encoding N-terminal fragment of lacZ α as a negative control. ( B ) E. coli ΔcysMΔcysK competent cells transformed with positive control c ysM , two cysM variants cysM-C / cysM-CM in pUC19 plasmid, and original pUC19 encoding N-terminal fragment of lacZ α as a negative control. Cells were plated on M9 + glucose medium with 0.4 mM IPTG, 50 μg/ml kanamycin, and 100 μg/ml ampicillin and incubated at 30 °C for 72 h.

    Techniques Used: Transformation Assay, Positive Control, Clone Assay, Plasmid Preparation, Negative Control, Incubation

    Growth curve of re-transformed auxotrophic E. coli strains in LB and M9 + glucose media. Each panel represents growth of cysteine-dependent E. coli auxotrophs rescued by wild type enzymes: CysE or CysM (blue), cysteine-free enzymes: CysE-C or CysM-C (orange), cysteine- and methionine-free enzymes: CysE-CM or CysM-CM (yellow), and LacZα protein expressed from original pUC19 plasmid (gray). Growth curve was monitored every 10 minutes at OD 600 nm using 96-well plate reader. Standard deviations of the growth curves are displayed as calculated from triplicates.
    Figure Legend Snippet: Growth curve of re-transformed auxotrophic E. coli strains in LB and M9 + glucose media. Each panel represents growth of cysteine-dependent E. coli auxotrophs rescued by wild type enzymes: CysE or CysM (blue), cysteine-free enzymes: CysE-C or CysM-C (orange), cysteine- and methionine-free enzymes: CysE-CM or CysM-CM (yellow), and LacZα protein expressed from original pUC19 plasmid (gray). Growth curve was monitored every 10 minutes at OD 600 nm using 96-well plate reader. Standard deviations of the growth curves are displayed as calculated from triplicates.

    Techniques Used: Transformation Assay, Plasmid Preparation

    22) Product Images from "Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4"

    Article Title: Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1191

    gp17 nuclease prefers long DNA substrates and cleaves at the ends of linear DNA. ( A ) Increasing concentrations of gp17 were incubated with 0.9 nM each of 29 kb pAd10 plasmid DNA or 2.6 kb pUC19 plasmid DNA. The undigested circular DNA was quantified and used to determine the percent of cleaved DNA at different gp17:DNA ratios. Values represent the average of duplicates from two independent experiments. ( B ) gp17 preference for longer DNA molecules was seen by incubating gp17 (3 µM, lanes 2–7) with a 2-log DNA ladder (400 ng, 0.1–10 kb, New England Biolabs) for 2–30 min. ( C ) Autoradiogram showing the cleavage of γ 32 P end-labeled λ-HindIII DNA fragments (0.5 pmol, 125–23 130 bp, Promega) by gp17 (1.2 µM) (lanes 2–6) or DNase I (0.0024 µM, 500-fold less than gp17) (lanes 7–11). Lane 1 has untreated DNA. ( D ) gp17 nuclease generates blunt ends. Circular pUC19 DNA (40 ng) was cleaved by gp17 (lanes 2–4) or BamH1 (lanes 5–7). The cleaved DNA was then treated with E. coli DNA ligase (lanes 3 and 6) or T4 DNA ligase (lanes 4 and 7). Lanes labeled as ‘C’ are control untreated lanes. See ‘Materials and Methods’ section for additional details.
    Figure Legend Snippet: gp17 nuclease prefers long DNA substrates and cleaves at the ends of linear DNA. ( A ) Increasing concentrations of gp17 were incubated with 0.9 nM each of 29 kb pAd10 plasmid DNA or 2.6 kb pUC19 plasmid DNA. The undigested circular DNA was quantified and used to determine the percent of cleaved DNA at different gp17:DNA ratios. Values represent the average of duplicates from two independent experiments. ( B ) gp17 preference for longer DNA molecules was seen by incubating gp17 (3 µM, lanes 2–7) with a 2-log DNA ladder (400 ng, 0.1–10 kb, New England Biolabs) for 2–30 min. ( C ) Autoradiogram showing the cleavage of γ 32 P end-labeled λ-HindIII DNA fragments (0.5 pmol, 125–23 130 bp, Promega) by gp17 (1.2 µM) (lanes 2–6) or DNase I (0.0024 µM, 500-fold less than gp17) (lanes 7–11). Lane 1 has untreated DNA. ( D ) gp17 nuclease generates blunt ends. Circular pUC19 DNA (40 ng) was cleaved by gp17 (lanes 2–4) or BamH1 (lanes 5–7). The cleaved DNA was then treated with E. coli DNA ligase (lanes 3 and 6) or T4 DNA ligase (lanes 4 and 7). Lanes labeled as ‘C’ are control untreated lanes. See ‘Materials and Methods’ section for additional details.

    Techniques Used: Incubation, Plasmid Preparation, Labeling

    23) Product Images from "A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage"

    Article Title: A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky806

    ( A ) BDNPP hydrolytic cleavage mechanism in the presence of Cu 2 TPNap; ( B ) Lineweaver–Burk plot; ( C ) rate-pH profile for the cleavage of BDNPP in the presence of Cu 2 TPNap at 40°C; ( D ) DNA cleavage reactions by Cu 2 TPNap on pUC19 plasmid DNA over 1 h at 37°C in the absence of added reductant; and ( E ) T4 DNA ligase experiments with Cu 2 TPNap and restriction enzymes EcoRI and Nt.BspQI.
    Figure Legend Snippet: ( A ) BDNPP hydrolytic cleavage mechanism in the presence of Cu 2 TPNap; ( B ) Lineweaver–Burk plot; ( C ) rate-pH profile for the cleavage of BDNPP in the presence of Cu 2 TPNap at 40°C; ( D ) DNA cleavage reactions by Cu 2 TPNap on pUC19 plasmid DNA over 1 h at 37°C in the absence of added reductant; and ( E ) T4 DNA ligase experiments with Cu 2 TPNap and restriction enzymes EcoRI and Nt.BspQI.

    Techniques Used: Plasmid Preparation

    ( A ) Cu 2 TPNap DNA cleavage experiments in the absence (lane 2–5) and presence of free radical antioxidants including DMSO ( • OH, lane 6–9), tiron (O 2 •− , lane 10–13), pyruvate (H 2 O 2 , lane 14–17) and sodium azide ( 1 O 2 , lane 18–21); ( B ) quantification of 8-oxo-dG lesions in pUC19 treated with 40 and 60 μM of Cu 2 )) and ( C ) M13mp18 single stranded plasmid DNA incubated with increasing concentrations of Cu 2 TPNap for 30 min at 37°C in the absence of added oxidant or reductant.
    Figure Legend Snippet: ( A ) Cu 2 TPNap DNA cleavage experiments in the absence (lane 2–5) and presence of free radical antioxidants including DMSO ( • OH, lane 6–9), tiron (O 2 •− , lane 10–13), pyruvate (H 2 O 2 , lane 14–17) and sodium azide ( 1 O 2 , lane 18–21); ( B ) quantification of 8-oxo-dG lesions in pUC19 treated with 40 and 60 μM of Cu 2 )) and ( C ) M13mp18 single stranded plasmid DNA incubated with increasing concentrations of Cu 2 TPNap for 30 min at 37°C in the absence of added oxidant or reductant.

    Techniques Used: Plasmid Preparation, Incubation

    24) Product Images from "Role for FimH in Extraintestinal Pathogenic Escherichia coli Invasion and Translocation through the Intestinal Epithelium"

    Article Title: Role for FimH in Extraintestinal Pathogenic Escherichia coli Invasion and Translocation through the Intestinal Epithelium

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00581-17

    FimH mediates ExPEC invasion of human jejunal intestinal enteroid monolayers (HJIEMs). HJIEMs were infected at an MOI of 100 with ExPEC wild-type (Wt) or Δ fimH strain or pUC19:Δ fimH ExPEC strain CP9, and adherence (A) and invasion (B) were measured (see Materials and Methods). (C) The effects of mannose on wild-type (Wt) ExPEC and Δ fimH mutant strains were evaluated as described in Materials and Methods. Data are means ± SEM; n = 4 to 8, assayed in triplicate; *, P ≤ 0.05.
    Figure Legend Snippet: FimH mediates ExPEC invasion of human jejunal intestinal enteroid monolayers (HJIEMs). HJIEMs were infected at an MOI of 100 with ExPEC wild-type (Wt) or Δ fimH strain or pUC19:Δ fimH ExPEC strain CP9, and adherence (A) and invasion (B) were measured (see Materials and Methods). (C) The effects of mannose on wild-type (Wt) ExPEC and Δ fimH mutant strains were evaluated as described in Materials and Methods. Data are means ± SEM; n = 4 to 8, assayed in triplicate; *, P ≤ 0.05.

    Techniques Used: Infection, Mutagenesis

    FimH is important for ExPEC translocation of an immunocompromised host. (A to G) BALB/c mice were subjected to oral gavage with wild-type (Wt), fimH mutant, or complemented pUC19:Δ fimH ExPEC strains at 10 9 CFU/100 μl. Mice were given cyclophosphamide at a total dose of 450 mg/kg (three 150-mg/kg doses on days 1, 3, and 5) by intraperitoneal injection. Intestinal colonization was measured by fecal homogenates (days 1, 3, 5, and 7) (A) or intestinal homogenates (8 days postinfection immediately after euthanasia) (B, C) plated on LB/agar plus 10 μg/ml CM. (D to G) Translocation out of the GI tract was determined by the presence of bacteria in organs as described in Materials and Methods. Data are means ± SEM; n = 14 to 16 for each group. *, P ≤ 0.05; **, P ≤ 0.01. (H) For an intraperitoneal infection, BALB/c mice were given 10 7 CFU/100 μl of wild-type (Wt) ExPEC or fimH mutant strains. Colonization was measured by organ homogenates plated on LB/agar plus 10 μg/ml CM (see Materials and Methods). Data are means ± SEM; n = 8 for each group.
    Figure Legend Snippet: FimH is important for ExPEC translocation of an immunocompromised host. (A to G) BALB/c mice were subjected to oral gavage with wild-type (Wt), fimH mutant, or complemented pUC19:Δ fimH ExPEC strains at 10 9 CFU/100 μl. Mice were given cyclophosphamide at a total dose of 450 mg/kg (three 150-mg/kg doses on days 1, 3, and 5) by intraperitoneal injection. Intestinal colonization was measured by fecal homogenates (days 1, 3, 5, and 7) (A) or intestinal homogenates (8 days postinfection immediately after euthanasia) (B, C) plated on LB/agar plus 10 μg/ml CM. (D to G) Translocation out of the GI tract was determined by the presence of bacteria in organs as described in Materials and Methods. Data are means ± SEM; n = 14 to 16 for each group. *, P ≤ 0.05; **, P ≤ 0.01. (H) For an intraperitoneal infection, BALB/c mice were given 10 7 CFU/100 μl of wild-type (Wt) ExPEC or fimH mutant strains. Colonization was measured by organ homogenates plated on LB/agar plus 10 μg/ml CM (see Materials and Methods). Data are means ± SEM; n = 8 for each group.

    Techniques Used: Translocation Assay, Mouse Assay, Mutagenesis, Injection, Infection

    25) Product Images from "C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents"

    Article Title: C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1097

    ( A ) Influence of ionic strength on pUC19 condensation (400 ng) by OC3 and MC3 (25 μM) opioid compounds. Condensation reactions on pUC19 (400 ng) by opioid compounds in ( B ) acidic NaOAc buffer (80 mM, pH = 4.0), and ( C ) basic Tris buffer (80 mM, pH = 9.0) in the presence of 25 mM NaCl.
    Figure Legend Snippet: ( A ) Influence of ionic strength on pUC19 condensation (400 ng) by OC3 and MC3 (25 μM) opioid compounds. Condensation reactions on pUC19 (400 ng) by opioid compounds in ( B ) acidic NaOAc buffer (80 mM, pH = 4.0), and ( C ) basic Tris buffer (80 mM, pH = 9.0) in the presence of 25 mM NaCl.

    Techniques Used:

    ( A ) Agarose gel electrophoresis of supercoiled (400 ng) and ( B ) a 742 bp dsDNA fragment of pUC19 (400 ng) exposed to increasing concentrations of MC3, OC3 and HC3 . Reactions were carried out in the presence of 25 mM NaCl for 5 h at 37°C prior to electrophoretic analysis.
    Figure Legend Snippet: ( A ) Agarose gel electrophoresis of supercoiled (400 ng) and ( B ) a 742 bp dsDNA fragment of pUC19 (400 ng) exposed to increasing concentrations of MC3, OC3 and HC3 . Reactions were carried out in the presence of 25 mM NaCl for 5 h at 37°C prior to electrophoretic analysis.

    Techniques Used: Agarose Gel Electrophoresis

    Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .
    Figure Legend Snippet: Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .

    Techniques Used: Microscopy

    26) Product Images from "The dual role of HOP2 in mammalian meiotic homologous recombination"

    Article Title: The dual role of HOP2 in mammalian meiotic homologous recombination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1234

    The mechanism of recombination mediated by HOP2. ( A ) Formation of joint molecules promoted by HOP2. (Panel a) The presynaptic polymerization of HOP2 protein on ssDNA. (Panel b) Conjunction of ssDNA and dsDNA without homologous alignment. (Panel c) Homologous DNA pairing and strand invasion. Deproteinization of the complex in panel (c) in the context of a supercoiled target duplex DNA results in a stable D-loop. ( B ) HOP2 unstacks bases upon binding to ssDNA. Unstacking is manifested as increased reactivity of the thymine residues to potassium permanganate (arrows). Lane 1 contains a ladder generated by cleavage at purine bases ( C ) Experimental outline for DNA capture assay. Biotinylated DNA, immobilized on streptavidin–agarose beads, was used as a binding substrate for HOP2 protein. After incubation with pUC19 dsDNA, nucleoprotein complexes were separated from unbound proteins and dsDNA by centrifugation, and products were deproteinized before analysis by gel electrophoresis. ( D ) Quantitation of captured DNA as determined by intensity of ethidium bromide fluorescence. ( E ) Schematic of the synaptic complex assay. Fluorescein and rhodamine are represented in green and red, respectively. ( F ) Synaptic complex formation promoted by HOP2 and DMC1 with homologous and heterologous DNA. Synaptic complexes for homologous dsDNA in the absence of protein and DMC1 with homologous DNA are also shown.
    Figure Legend Snippet: The mechanism of recombination mediated by HOP2. ( A ) Formation of joint molecules promoted by HOP2. (Panel a) The presynaptic polymerization of HOP2 protein on ssDNA. (Panel b) Conjunction of ssDNA and dsDNA without homologous alignment. (Panel c) Homologous DNA pairing and strand invasion. Deproteinization of the complex in panel (c) in the context of a supercoiled target duplex DNA results in a stable D-loop. ( B ) HOP2 unstacks bases upon binding to ssDNA. Unstacking is manifested as increased reactivity of the thymine residues to potassium permanganate (arrows). Lane 1 contains a ladder generated by cleavage at purine bases ( C ) Experimental outline for DNA capture assay. Biotinylated DNA, immobilized on streptavidin–agarose beads, was used as a binding substrate for HOP2 protein. After incubation with pUC19 dsDNA, nucleoprotein complexes were separated from unbound proteins and dsDNA by centrifugation, and products were deproteinized before analysis by gel electrophoresis. ( D ) Quantitation of captured DNA as determined by intensity of ethidium bromide fluorescence. ( E ) Schematic of the synaptic complex assay. Fluorescein and rhodamine are represented in green and red, respectively. ( F ) Synaptic complex formation promoted by HOP2 and DMC1 with homologous and heterologous DNA. Synaptic complexes for homologous dsDNA in the absence of protein and DMC1 with homologous DNA are also shown.

    Techniques Used: Binding Assay, Generated, Incubation, Centrifugation, Nucleic Acid Electrophoresis, Quantitation Assay, Fluorescence

    27) Product Images from "A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage"

    Article Title: A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky806

    ( A ) BDNPP hydrolytic cleavage mechanism in the presence of Cu 2 TPNap; ( B ) Lineweaver–Burk plot; ( C ) rate-pH profile for the cleavage of BDNPP in the presence of Cu 2 TPNap at 40°C; ( D ) DNA cleavage reactions by Cu 2 TPNap on pUC19 plasmid DNA over 1 h at 37°C in the absence of added reductant; and ( E ) T4 DNA ligase experiments with Cu 2 TPNap and restriction enzymes EcoRI and Nt.BspQI.
    Figure Legend Snippet: ( A ) BDNPP hydrolytic cleavage mechanism in the presence of Cu 2 TPNap; ( B ) Lineweaver–Burk plot; ( C ) rate-pH profile for the cleavage of BDNPP in the presence of Cu 2 TPNap at 40°C; ( D ) DNA cleavage reactions by Cu 2 TPNap on pUC19 plasmid DNA over 1 h at 37°C in the absence of added reductant; and ( E ) T4 DNA ligase experiments with Cu 2 TPNap and restriction enzymes EcoRI and Nt.BspQI.

    Techniques Used: Plasmid Preparation

    ( A ) Cu 2 TPNap DNA cleavage experiments in the absence (lane 2–5) and presence of free radical antioxidants including DMSO ( • OH, lane 6–9), tiron (O 2 •− , lane 10–13), pyruvate (H 2 O 2 , lane 14–17) and sodium azide ( 1 O 2 , lane 18–21); ( B ) quantification of 8-oxo-dG lesions in pUC19 treated with 40 and 60 μM of Cu 2 TPNap for 4 h at 37°C and compared directly to Cu-Phen and Cu-Terph (reported elsewhere, ( 44 )) and ( C ) M13mp18 single stranded plasmid DNA incubated with increasing concentrations of Cu 2 TPNap for 30 min at 37°C in the absence of added oxidant or reductant.
    Figure Legend Snippet: ( A ) Cu 2 TPNap DNA cleavage experiments in the absence (lane 2–5) and presence of free radical antioxidants including DMSO ( • OH, lane 6–9), tiron (O 2 •− , lane 10–13), pyruvate (H 2 O 2 , lane 14–17) and sodium azide ( 1 O 2 , lane 18–21); ( B ) quantification of 8-oxo-dG lesions in pUC19 treated with 40 and 60 μM of Cu 2 TPNap for 4 h at 37°C and compared directly to Cu-Phen and Cu-Terph (reported elsewhere, ( 44 )) and ( C ) M13mp18 single stranded plasmid DNA incubated with increasing concentrations of Cu 2 TPNap for 30 min at 37°C in the absence of added oxidant or reductant.

    Techniques Used: Plasmid Preparation, Incubation

    28) Product Images from "Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel"

    Article Title: Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1117549109

    An epigenetic analysis workflow using fluorescence-activated, single molecule sorting. ( Top ) DNA preparation. Linearized DNA plasmids, unmethylated pUC19, and methylated pML4.2 were fluorescently labeled with a red stain and then mixed with green-labeled
    Figure Legend Snippet: An epigenetic analysis workflow using fluorescence-activated, single molecule sorting. ( Top ) DNA preparation. Linearized DNA plasmids, unmethylated pUC19, and methylated pML4.2 were fluorescently labeled with a red stain and then mixed with green-labeled

    Techniques Used: Fluorescence, Methylation, Labeling, Staining

    Single molecule detection and sorting of methylated DNA. DNA methylation state was identified using a green-labeled methyl binding domain-1 (MBD1) protein incubated in a mixture of red-labeled DNAs including unmethylated-pUC19 and methylated-pML4.2. Methylation-specific
    Figure Legend Snippet: Single molecule detection and sorting of methylated DNA. DNA methylation state was identified using a green-labeled methyl binding domain-1 (MBD1) protein incubated in a mixture of red-labeled DNAs including unmethylated-pUC19 and methylated-pML4.2. Methylation-specific

    Techniques Used: Methylation, DNA Methylation Assay, Labeling, Binding Assay, Incubation

    Optimization of molecule sorting efficiency. A 1∶4 molar ratio mixture of red-intercalated pML4.2 (15.2 Kb) and pUC19 (2.7 Kb) DNA was loaded into the nanofluidic device and sorted on differences in fluorescence intensity to collect
    Figure Legend Snippet: Optimization of molecule sorting efficiency. A 1∶4 molar ratio mixture of red-intercalated pML4.2 (15.2 Kb) and pUC19 (2.7 Kb) DNA was loaded into the nanofluidic device and sorted on differences in fluorescence intensity to collect

    Techniques Used: Fluorescence

    29) Product Images from "Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer"

    Article Title: Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00787-16

    In vitro analysis of RpnA endonuclease activity. (A) WT RpnA cleaves pUC19, RpnA-D63A does not cleave pUC19, and RpnA-D165A is more active on pUC19. The pUC19 DNA (29 nM, 50 μg/ml) is initially supercoiled but can be relaxed by nicks, linearized by double-strand cleavage, or cleaved further. The supercoiled (control), relaxed (Nb.BtsI), and linear (HindIII) forms are indicated. pUC19 was treated with RpnA-inactive RpnA-D63A or hyperactive RpnA-D165A (15 μM, 45 min). (B) Time course of an RpnA (7.5 μM)-pUC19 (29 nM) digest. Band intensity was compared to determine the relative amounts of supercoiled, nicked, and linear pUC19 at each time point. Over 90% of the supercoiled pUC19 was digested within 180 min. (C) RpnA endonuclease activity depends on divalent cation and is stimulated by Ca 2+ . The reaction buffer was 50 mM NaCl and 10 mM Tris, pH 8.0; the indicated additives were at 10 mM each. RpnA at 3.8 μM was added for 18 h. (D) RpnA cleavage products provide a DNA polymerase primer. pUC19 was digested with RpnA, DNase I, or micrococcal nuclease (MNase) to produce similar smears and then incubated with fluorescein-labeled dNTPs and the Klenow fragment of DNA polymerase. DNA was visualized by ethidium bromide (EtBr; left) or fluorescein (middle), with the two signals being merged at the right. RpnA- and DNase I-digested DNAs were effectively labeled, but micrococcal nuclease-digested DNA was not.
    Figure Legend Snippet: In vitro analysis of RpnA endonuclease activity. (A) WT RpnA cleaves pUC19, RpnA-D63A does not cleave pUC19, and RpnA-D165A is more active on pUC19. The pUC19 DNA (29 nM, 50 μg/ml) is initially supercoiled but can be relaxed by nicks, linearized by double-strand cleavage, or cleaved further. The supercoiled (control), relaxed (Nb.BtsI), and linear (HindIII) forms are indicated. pUC19 was treated with RpnA-inactive RpnA-D63A or hyperactive RpnA-D165A (15 μM, 45 min). (B) Time course of an RpnA (7.5 μM)-pUC19 (29 nM) digest. Band intensity was compared to determine the relative amounts of supercoiled, nicked, and linear pUC19 at each time point. Over 90% of the supercoiled pUC19 was digested within 180 min. (C) RpnA endonuclease activity depends on divalent cation and is stimulated by Ca 2+ . The reaction buffer was 50 mM NaCl and 10 mM Tris, pH 8.0; the indicated additives were at 10 mM each. RpnA at 3.8 μM was added for 18 h. (D) RpnA cleavage products provide a DNA polymerase primer. pUC19 was digested with RpnA, DNase I, or micrococcal nuclease (MNase) to produce similar smears and then incubated with fluorescein-labeled dNTPs and the Klenow fragment of DNA polymerase. DNA was visualized by ethidium bromide (EtBr; left) or fluorescein (middle), with the two signals being merged at the right. RpnA- and DNase I-digested DNAs were effectively labeled, but micrococcal nuclease-digested DNA was not.

    Techniques Used: In Vitro, Activity Assay, Incubation, Labeling

    30) Product Images from "Identification of the first eubacterial endonuclease coded by an intein allele in the pps1 gene of mycobacteria"

    Article Title: Identification of the first eubacterial endonuclease coded by an intein allele in the pps1 gene of mycobacteria

    Journal: Nucleic Acids Research

    doi:

    Cleavage assay for Mga Pps1. Aliquots of 100 ng Sca I-linearized p Mga Site substrate ( A ) or linearized pUC19 ( B ) were incubated with 250 ng of a crude extract of (lane 1) non-transformed BL21(DE3)[pLysS], (lane 2) untagged Mga Pps1 or (lane 3) tagged Mga Pps1, or with (lane 4) 100, (lane 5) 50 or (lane 6) 25 ng of a crude extract of untagged Mga Pps1 for 10 min at 37°C, or with 25 ng of a crude extract of untagged Mga Pps1 for (lanes 7–12) 0, 10, 30, 40, 100 or 150 min, in 10 mM Tris–HCl, pH 8, buffer containing 10 mM MgCl 2 and 25 mM KCl.
    Figure Legend Snippet: Cleavage assay for Mga Pps1. Aliquots of 100 ng Sca I-linearized p Mga Site substrate ( A ) or linearized pUC19 ( B ) were incubated with 250 ng of a crude extract of (lane 1) non-transformed BL21(DE3)[pLysS], (lane 2) untagged Mga Pps1 or (lane 3) tagged Mga Pps1, or with (lane 4) 100, (lane 5) 50 or (lane 6) 25 ng of a crude extract of untagged Mga Pps1 for 10 min at 37°C, or with 25 ng of a crude extract of untagged Mga Pps1 for (lanes 7–12) 0, 10, 30, 40, 100 or 150 min, in 10 mM Tris–HCl, pH 8, buffer containing 10 mM MgCl 2 and 25 mM KCl.

    Techniques Used: Cleavage Assay, Incubation, Transformation Assay

    31) Product Images from "The Hyperthermophilic Restriction-Modification Systems of Thermococcus kodakarensis Protect Genome Integrity"

    Article Title: The Hyperthermophilic Restriction-Modification Systems of Thermococcus kodakarensis Protect Genome Integrity

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2021.657356

    Characterization of TkoI restriction endonuclease activities. (A) Predicted TkoI recognition sites in plasmid pBR322 are at positions 1,549, 2,040, and 3,220. (B) TkoI was incubated with pBR322 in 1X NEBuffer 3 supplemented with SAM and 90mer trans-DNA for 30 min at 65°C. Reactions were halted with Proteinase K and loading dye and separated on a 1% agarose gel. Lane 1 is a 1 kb DNA ladder. Lane 2 is pBR322 alone and lanes 3–7 are decreasing amounts of TkoI (400–25 nM). (C) TkoI cleaves pUC19 at a single site. (D) pUC19/TkoI cut fragments were analyzed by run off sequencing to determine TkoI cut sites on the top and bottom strands: GTGAAG(N) 20 /(N) 18 .
    Figure Legend Snippet: Characterization of TkoI restriction endonuclease activities. (A) Predicted TkoI recognition sites in plasmid pBR322 are at positions 1,549, 2,040, and 3,220. (B) TkoI was incubated with pBR322 in 1X NEBuffer 3 supplemented with SAM and 90mer trans-DNA for 30 min at 65°C. Reactions were halted with Proteinase K and loading dye and separated on a 1% agarose gel. Lane 1 is a 1 kb DNA ladder. Lane 2 is pBR322 alone and lanes 3–7 are decreasing amounts of TkoI (400–25 nM). (C) TkoI cleaves pUC19 at a single site. (D) pUC19/TkoI cut fragments were analyzed by run off sequencing to determine TkoI cut sites on the top and bottom strands: GTGAAG(N) 20 /(N) 18 .

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Sequencing

    Characterization of TkoII restriction endonuclease activities. (A) Predicted TkoII recognition sites in plasmid pBR322 are at positions 1093, 2944 and 4355. (B) TkoII was incubated with pBR322 in 1X NEBuffer 1 supplemented with SAM and 90mer trans-DNA for 30 min at 65°C. Reactions were halted with Proteinase K and loading dye and separated on a 1% agarose gel. Lane 1 is a 1 kb DNA ladder. Lane 2 is pBR322 alone and lanes 3–7 are decreasing amounts of TkoII (10–0.62 nM). (C) TkoII cleaves pUC19 at a single site. (D) pUC19/TkoII cut fragments were analyzed by run off sequencing to determine TkoII cut sites on the top and bottom strands: TTCAAG(N) 10 /(N) 8 .
    Figure Legend Snippet: Characterization of TkoII restriction endonuclease activities. (A) Predicted TkoII recognition sites in plasmid pBR322 are at positions 1093, 2944 and 4355. (B) TkoII was incubated with pBR322 in 1X NEBuffer 1 supplemented with SAM and 90mer trans-DNA for 30 min at 65°C. Reactions were halted with Proteinase K and loading dye and separated on a 1% agarose gel. Lane 1 is a 1 kb DNA ladder. Lane 2 is pBR322 alone and lanes 3–7 are decreasing amounts of TkoII (10–0.62 nM). (C) TkoII cleaves pUC19 at a single site. (D) pUC19/TkoII cut fragments were analyzed by run off sequencing to determine TkoII cut sites on the top and bottom strands: TTCAAG(N) 10 /(N) 8 .

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Sequencing

    32) Product Images from "Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants"

    Article Title: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkj483

    Variant E156K digestion of a substrate containing a single 5′-GCTGCCGC-3′ site. Plasmid pUC-GCT was derived from pUC19. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer.
    Figure Legend Snippet: Variant E156K digestion of a substrate containing a single 5′-GCTGCCGC-3′ site. Plasmid pUC-GCT was derived from pUC19. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer.

    Techniques Used: Variant Assay, Plasmid Preparation, Derivative Assay, Incubation

    33) Product Images from "Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes"

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-19920-y

    Synthetic c ysE and cysM gene transformants display recovery of CysE function without cysteine and methionine and CysM function without cysteine. ( A ) E. coli ΔcysE competent cells were transformed with positive control cysE , two cysE variants cysE-C/cysE-CM cloned into the multiple cloning site of pUC19 plasmid, and original pUC19 encoding N-terminal fragment of lacZ α as a negative control. ( B ) E. coli ΔcysMΔcysK competent cells transformed with positive control c ysM , two cysM variants cysM-C / cysM-CM in pUC19 plasmid, and original pUC19 encoding N-terminal fragment of lacZ α as a negative control. Cells were plated on M9 + glucose medium with 0.4 mM IPTG, 50 μg/ml kanamycin, and 100 μg/ml ampicillin and incubated at 30 °C for 72 h.
    Figure Legend Snippet: Synthetic c ysE and cysM gene transformants display recovery of CysE function without cysteine and methionine and CysM function without cysteine. ( A ) E. coli ΔcysE competent cells were transformed with positive control cysE , two cysE variants cysE-C/cysE-CM cloned into the multiple cloning site of pUC19 plasmid, and original pUC19 encoding N-terminal fragment of lacZ α as a negative control. ( B ) E. coli ΔcysMΔcysK competent cells transformed with positive control c ysM , two cysM variants cysM-C / cysM-CM in pUC19 plasmid, and original pUC19 encoding N-terminal fragment of lacZ α as a negative control. Cells were plated on M9 + glucose medium with 0.4 mM IPTG, 50 μg/ml kanamycin, and 100 μg/ml ampicillin and incubated at 30 °C for 72 h.

    Techniques Used: Transformation Assay, Positive Control, Clone Assay, Plasmid Preparation, Negative Control, Incubation

    34) Product Images from "C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents"

    Article Title: C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1097

    ( A ) Influence of ionic strength on pUC19 condensation (400 ng) by OC3 and MC3 (25 μM) opioid compounds. Condensation reactions on pUC19 (400 ng) by opioid compounds in ( B ) acidic NaOAc buffer (80 mM, pH = 4.0), and ( C ) basic Tris buffer (80 mM, pH = 9.0) in the presence of 25 mM NaCl.
    Figure Legend Snippet: ( A ) Influence of ionic strength on pUC19 condensation (400 ng) by OC3 and MC3 (25 μM) opioid compounds. Condensation reactions on pUC19 (400 ng) by opioid compounds in ( B ) acidic NaOAc buffer (80 mM, pH = 4.0), and ( C ) basic Tris buffer (80 mM, pH = 9.0) in the presence of 25 mM NaCl.

    Techniques Used:

    ( A ) Agarose gel electrophoresis of supercoiled (400 ng) and ( B ) a 742 bp dsDNA fragment of pUC19 (400 ng) exposed to increasing concentrations of MC3, OC3 and HC3 . Reactions were carried out in the presence of 25 mM NaCl for 5 h at 37°C prior to electrophoretic analysis.
    Figure Legend Snippet: ( A ) Agarose gel electrophoresis of supercoiled (400 ng) and ( B ) a 742 bp dsDNA fragment of pUC19 (400 ng) exposed to increasing concentrations of MC3, OC3 and HC3 . Reactions were carried out in the presence of 25 mM NaCl for 5 h at 37°C prior to electrophoretic analysis.

    Techniques Used: Agarose Gel Electrophoresis

    Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .
    Figure Legend Snippet: Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .

    Techniques Used: Microscopy

    35) Product Images from "Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants"

    Article Title: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkj483

    Variant E156K digestion of a substrate containing a single 5′-GCTGCCGC-3′ site. Plasmid pUC-GCT was derived from pUC19. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer.
    Figure Legend Snippet: Variant E156K digestion of a substrate containing a single 5′-GCTGCCGC-3′ site. Plasmid pUC-GCT was derived from pUC19. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer.

    Techniques Used: Variant Assay, Plasmid Preparation, Derivative Assay, Incubation

    36) Product Images from "Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974 ▿"

    Article Title: Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974 ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00782-09

    In vivo activities of Streptomyces SerRS1 and SerRS2. s erS1 , s erS2 , E. coli s erS , and the s erS2 ( H270G ) mutant were cloned into pUC19 (pUC) vector under the control of the glnS ). (A) The plasmids were transformed into an E. coli temperature-sensitive
    Figure Legend Snippet: In vivo activities of Streptomyces SerRS1 and SerRS2. s erS1 , s erS2 , E. coli s erS , and the s erS2 ( H270G ) mutant were cloned into pUC19 (pUC) vector under the control of the glnS ). (A) The plasmids were transformed into an E. coli temperature-sensitive

    Techniques Used: In Vivo, Mutagenesis, Clone Assay, Plasmid Preparation, Transformation Assay

    37) Product Images from "Secretory expression of biologically active human Herpes virus interleukin-10 analogues in Escherichia coli via a modified Sec-dependent transporter construct"

    Article Title: Secretory expression of biologically active human Herpes virus interleukin-10 analogues in Escherichia coli via a modified Sec-dependent transporter construct

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-13-82

    Immunoblot analysis of viral IL-10 recombinant proteins. E. coli BL21 (DE3) derived recombinant viral IL-10 proteins were analyzed by immunoblot in different concentrations and from different cell compartments under both reducing ( A = HCMV IL-10; C = EBV IL-10) and non-reducing conditions ( B = HCMV IL-10). Periplasmic and cytoplasmic fractions of pUC19 transformed E. coli BL21 (DE3) cells and commercial viral IL-10 proteins served as controls. One experiment representative of three is shown. SN = supernatant; PP = periplasmic fraction; CP = cytoplasmic fraction; Com. = commercial; Rec. = recombinant.
    Figure Legend Snippet: Immunoblot analysis of viral IL-10 recombinant proteins. E. coli BL21 (DE3) derived recombinant viral IL-10 proteins were analyzed by immunoblot in different concentrations and from different cell compartments under both reducing ( A = HCMV IL-10; C = EBV IL-10) and non-reducing conditions ( B = HCMV IL-10). Periplasmic and cytoplasmic fractions of pUC19 transformed E. coli BL21 (DE3) cells and commercial viral IL-10 proteins served as controls. One experiment representative of three is shown. SN = supernatant; PP = periplasmic fraction; CP = cytoplasmic fraction; Com. = commercial; Rec. = recombinant.

    Techniques Used: Recombinant, Derivative Assay, Transformation Assay

    Plasmid maps of HCMV IL-10 (pAZ1c; A) and EBV (pGA6; B) expression vectors are depicted. The artificial transporter consists of the E. coli ompF signal sequence fused in frame to E. coli codon optimized mature viral IL-10 genes under control of the T7 promoter. For subcloning, the constructs are flanked by Eco RI restriction sites. Plasmid pGA6 contains a ColE1, pAZ1c a pUC19-derived pMB1 origin of replication.
    Figure Legend Snippet: Plasmid maps of HCMV IL-10 (pAZ1c; A) and EBV (pGA6; B) expression vectors are depicted. The artificial transporter consists of the E. coli ompF signal sequence fused in frame to E. coli codon optimized mature viral IL-10 genes under control of the T7 promoter. For subcloning, the constructs are flanked by Eco RI restriction sites. Plasmid pGA6 contains a ColE1, pAZ1c a pUC19-derived pMB1 origin of replication.

    Techniques Used: Plasmid Preparation, Expressing, Sequencing, Subcloning, Construct, Derivative Assay

    Activation of STAT3 by E. coli derived viral IL-10. STAT3 phosphorylation (STAT3-pY705) was analyzed by immunoblot of protein extracts from human Daudi cells (HCMV IL-10; A) and J774.1 mouse macrophages (EBV IL-10; B) treated with different concentrations of bacteria-derived viral IL-10. Total STAT3 was used to ensure equal protein loading in all lanes (double bands in Daudi cells represent STAT3 isoforms α and β). Periplasmic and cytoplasmic fractions of pUC19 transformed E. coli BL21 (DE3) cells and commercial viral IL-10 proteins served as controls. One experiment representative of two is shown. PP = periplasmic fraction; CP = cytoplasmic fraction; Com. = commercial; Rec. = recombinant.
    Figure Legend Snippet: Activation of STAT3 by E. coli derived viral IL-10. STAT3 phosphorylation (STAT3-pY705) was analyzed by immunoblot of protein extracts from human Daudi cells (HCMV IL-10; A) and J774.1 mouse macrophages (EBV IL-10; B) treated with different concentrations of bacteria-derived viral IL-10. Total STAT3 was used to ensure equal protein loading in all lanes (double bands in Daudi cells represent STAT3 isoforms α and β). Periplasmic and cytoplasmic fractions of pUC19 transformed E. coli BL21 (DE3) cells and commercial viral IL-10 proteins served as controls. One experiment representative of two is shown. PP = periplasmic fraction; CP = cytoplasmic fraction; Com. = commercial; Rec. = recombinant.

    Techniques Used: Activation Assay, Derivative Assay, Transformation Assay, Recombinant

    Inhibition of LPS-induced TNF-α release by E. coli derived recombinant EBV IL-10. J774.1 mouse macrophages were incubated with E. coli BL21 (DE3) pGA6 periplasmic fraction alone (bacterial recombinant EBV IL-10 at ~ 400 ng/ml) or in the presence of neutralizing monoclonal anti-EBV IL-10 antibody. Periplasmic fractions of E. coli BL21 (DE3) pUC19 were used as TNF-α induction control. Commercial EBV IL-10 (at ~ 400 ng/ml) served as positive control. TNF-α induction levels were set at 100%, and changes of TNF-α release are the means ± SD of four independent experiments. Statistical significance was determined using the Student t-test. Asterisks indicate statistically significant differences (* p ≤ 0.05; ** p ≤ 0.01) between pGA6 PP, pUC19 PP, and pGA6 after anti-EBV IL-10 treatment. PP = periplasmic fraction; Com. = commercial; Rec. = recombinant; mAb = monoclonal antibody.
    Figure Legend Snippet: Inhibition of LPS-induced TNF-α release by E. coli derived recombinant EBV IL-10. J774.1 mouse macrophages were incubated with E. coli BL21 (DE3) pGA6 periplasmic fraction alone (bacterial recombinant EBV IL-10 at ~ 400 ng/ml) or in the presence of neutralizing monoclonal anti-EBV IL-10 antibody. Periplasmic fractions of E. coli BL21 (DE3) pUC19 were used as TNF-α induction control. Commercial EBV IL-10 (at ~ 400 ng/ml) served as positive control. TNF-α induction levels were set at 100%, and changes of TNF-α release are the means ± SD of four independent experiments. Statistical significance was determined using the Student t-test. Asterisks indicate statistically significant differences (* p ≤ 0.05; ** p ≤ 0.01) between pGA6 PP, pUC19 PP, and pGA6 after anti-EBV IL-10 treatment. PP = periplasmic fraction; Com. = commercial; Rec. = recombinant; mAb = monoclonal antibody.

    Techniques Used: Inhibition, Derivative Assay, Recombinant, Incubation, Positive Control

    38) Product Images from "C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents"

    Article Title: C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1097

    ( A ) Influence of ionic strength on pUC19 condensation (400 ng) by OC3 and MC3 (25 μM) opioid compounds. Condensation reactions on pUC19 (400 ng) by opioid compounds in ( B ) acidic NaOAc buffer (80 mM, pH = 4.0), and ( C ) basic Tris buffer (80 mM, pH = 9.0) in the presence of 25 mM NaCl.
    Figure Legend Snippet: ( A ) Influence of ionic strength on pUC19 condensation (400 ng) by OC3 and MC3 (25 μM) opioid compounds. Condensation reactions on pUC19 (400 ng) by opioid compounds in ( B ) acidic NaOAc buffer (80 mM, pH = 4.0), and ( C ) basic Tris buffer (80 mM, pH = 9.0) in the presence of 25 mM NaCl.

    Techniques Used:

    ( A ) Agarose gel electrophoresis of supercoiled (400 ng) and ( B ) a 742 bp dsDNA fragment of pUC19 (400 ng) exposed to increasing concentrations of MC3, OC3 and HC3 . Reactions were carried out in the presence of 25 mM NaCl for 5 h at 37°C prior to electrophoretic analysis.
    Figure Legend Snippet: ( A ) Agarose gel electrophoresis of supercoiled (400 ng) and ( B ) a 742 bp dsDNA fragment of pUC19 (400 ng) exposed to increasing concentrations of MC3, OC3 and HC3 . Reactions were carried out in the presence of 25 mM NaCl for 5 h at 37°C prior to electrophoretic analysis.

    Techniques Used: Agarose Gel Electrophoresis

    Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .
    Figure Legend Snippet: Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .

    Techniques Used: Microscopy

    39) Product Images from "Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants"

    Article Title: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkj483

    Variant E156K digestion of a substrate containing a single 5′-GCTGCCGC-3′ site. Plasmid pUC-GCT was derived from pUC19. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer.
    Figure Legend Snippet: Variant E156K digestion of a substrate containing a single 5′-GCTGCCGC-3′ site. Plasmid pUC-GCT was derived from pUC19. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer.

    Techniques Used: Variant Assay, Plasmid Preparation, Derivative Assay, Incubation

    40) Product Images from "Topological DNA-binding of structural maintenance of chromosomes-like RecN promotes DNA double-strand break repair in Escherichia coli"

    Article Title: Topological DNA-binding of structural maintenance of chromosomes-like RecN promotes DNA double-strand break repair in Escherichia coli

    Journal: Communications Biology

    doi: 10.1038/s42003-019-0655-4

    RecN promotes RecA-mediated D-loop formation and DNA strand exchange. a D-loop assays. RecA (2 µM) was incubated for 10 min in the presence of the pUC19-derivative linear ds/ssDNA (probe), and then RecN (1 µM) was added to the reaction mixture, followed by incubation for a further 15 min. The reactions were initiated by adding homologous pUC19 supercoiled circular DNA (target) and then incubated for another 5 min. The samples were analyzed by agarose gel electrophoresis and SYBR Gold staining. b Quantification of the amounts of D-loop products in the gel image shown in a . Data represent the mean ± standard deviation of three independent experiments. c Strand exchange assays. RecA (0.6 µM) was incubated for 10 min in the presence of phiX174 circular ssDNA (Css). The indicated concentrations (0–1.5 µM) of RecN were added to the reaction mixture, followed by incubation for 15 min. Subsequently, ssDNA-binding protein was added and samples were incubated for an additional 10 min. The reactions were initiated by adding homologous phiX174 linear dsDNA (Lds), incubated for 90 min, and then stopped by the addition of stop buffer. The samples were analyzed by agarose gel electrophoresis and SYBR Gold staining. d Quantification of the amounts of nicked circular dsDNA (NC) products in the gel image shown in c . Data represent the mean ± standard deviation of three independent experiments. e Time-course strand exchange experiments. RecA (0.8 µM) was incubated with or without RecN (1 µM). Aliquots were collected at the indicated time points and analyzed as described above. f Quantification of the amounts of NC DNA products in the gel image shown in e . c , e The arrowheads indicate the DNA substrates (Css and Lds) and the resulting products: joint molecules (JM), nicked circular dsDNA (NC), and linear ssDNA (Lss)
    Figure Legend Snippet: RecN promotes RecA-mediated D-loop formation and DNA strand exchange. a D-loop assays. RecA (2 µM) was incubated for 10 min in the presence of the pUC19-derivative linear ds/ssDNA (probe), and then RecN (1 µM) was added to the reaction mixture, followed by incubation for a further 15 min. The reactions were initiated by adding homologous pUC19 supercoiled circular DNA (target) and then incubated for another 5 min. The samples were analyzed by agarose gel electrophoresis and SYBR Gold staining. b Quantification of the amounts of D-loop products in the gel image shown in a . Data represent the mean ± standard deviation of three independent experiments. c Strand exchange assays. RecA (0.6 µM) was incubated for 10 min in the presence of phiX174 circular ssDNA (Css). The indicated concentrations (0–1.5 µM) of RecN were added to the reaction mixture, followed by incubation for 15 min. Subsequently, ssDNA-binding protein was added and samples were incubated for an additional 10 min. The reactions were initiated by adding homologous phiX174 linear dsDNA (Lds), incubated for 90 min, and then stopped by the addition of stop buffer. The samples were analyzed by agarose gel electrophoresis and SYBR Gold staining. d Quantification of the amounts of nicked circular dsDNA (NC) products in the gel image shown in c . Data represent the mean ± standard deviation of three independent experiments. e Time-course strand exchange experiments. RecA (0.8 µM) was incubated with or without RecN (1 µM). Aliquots were collected at the indicated time points and analyzed as described above. f Quantification of the amounts of NC DNA products in the gel image shown in e . c , e The arrowheads indicate the DNA substrates (Css and Lds) and the resulting products: joint molecules (JM), nicked circular dsDNA (NC), and linear ssDNA (Lss)

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Staining, Standard Deviation, Binding Assay

    RecA prevents the release of RecN from ssDNA ends. a Schematic illustration of His pull-down assays in b . b RecN (1 µM) was mixed with a linear ds/ssDNA (Lds/ss). The RecN-Lds/ss complex was collected using Co 2+ -conjugated beads, washed in buffer containing 50 mM KCl. To initiate the reactions, the bead suspension was incubated in the presence or absence of RecA (1 µM) at 37 °C. Aliquots were removed for analysis at the indicated time points. The bead-bound materials were recovered and analyzed by agarose gel electrophoresis (for DNA) and SDS–PAGE (for proteins). c The control experiment was performed in the absence of RecA as described for b , with the exception that pUC19 circular dsDNA (Cds) was used in place of Lds/ss. d Quantification of the band intensities of DNA substrates in the agarose gel images shown in b and c . The amount of DNA recovered at time zero was defined as 100%. Data represent the mean ± standard deviation of three independent experiments. e The physical interaction of RecN with RecA. The reaction mixtures were incubated in the presence of the indicated proteins (1 µM each protein). The proteins bound to Co 2+ -conjugated beads were collected, washed, eluted with SDS–sample buffer, and then analyzed by SDS–PAGE and CBB staining
    Figure Legend Snippet: RecA prevents the release of RecN from ssDNA ends. a Schematic illustration of His pull-down assays in b . b RecN (1 µM) was mixed with a linear ds/ssDNA (Lds/ss). The RecN-Lds/ss complex was collected using Co 2+ -conjugated beads, washed in buffer containing 50 mM KCl. To initiate the reactions, the bead suspension was incubated in the presence or absence of RecA (1 µM) at 37 °C. Aliquots were removed for analysis at the indicated time points. The bead-bound materials were recovered and analyzed by agarose gel electrophoresis (for DNA) and SDS–PAGE (for proteins). c The control experiment was performed in the absence of RecA as described for b , with the exception that pUC19 circular dsDNA (Cds) was used in place of Lds/ss. d Quantification of the band intensities of DNA substrates in the agarose gel images shown in b and c . The amount of DNA recovered at time zero was defined as 100%. Data represent the mean ± standard deviation of three independent experiments. e The physical interaction of RecN with RecA. The reaction mixtures were incubated in the presence of the indicated proteins (1 µM each protein). The proteins bound to Co 2+ -conjugated beads were collected, washed, eluted with SDS–sample buffer, and then analyzed by SDS–PAGE and CBB staining

    Techniques Used: Incubation, Agarose Gel Electrophoresis, SDS Page, Standard Deviation, Staining

    Related Articles

    other:

    Article Title: Depurination of colibactin-derived interstrand cross-links.
    Article Snippet: For each reaction with E.coli , 800 ng of linearized pUC19 DNA was added to 200 μL (6.2 μM base pairs) of M9-CA medium inoculated with 1.2 × 107 bacteria pre-grown to exponential phase in the M9-CA medium.

    Purification:

    Article Title: Methods and Techniques to Facilitate the Development of Clostridium novyi NT as an Effective, Therapeutic Oncolytic Bacteria
    Article Snippet: Transformation of Calcium Competent C. novyi Calcium competent C. novyi cells were allowed to thaw on ice. .. Subsequently, 5 μg of either purified pUC19 control plasmid (New England BioLabs Inc.) or the purified pKMD002 plasmid was added to an empty prechilled 15 mL tube (4°C). .. Competent C. novyi cells (100 μL) were added directly on top of the plasmid DNA in the prechilled tube without vortexing or mixing.

    Plasmid Preparation:

    Article Title: Methods and Techniques to Facilitate the Development of Clostridium novyi NT as an Effective, Therapeutic Oncolytic Bacteria
    Article Snippet: Transformation of Calcium Competent C. novyi Calcium competent C. novyi cells were allowed to thaw on ice. .. Subsequently, 5 μg of either purified pUC19 control plasmid (New England BioLabs Inc.) or the purified pKMD002 plasmid was added to an empty prechilled 15 mL tube (4°C). .. Competent C. novyi cells (100 μL) were added directly on top of the plasmid DNA in the prechilled tube without vortexing or mixing.

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
    Article Snippet: .. The 4mC control is generated by PCR amplification with 4mdCTP using part of the pUC19 vector (∼1 kb) as a template, whereas the C/5mC control is generated by treating unmethylated lambda DNA with the CpG methyltransferase (M. SssI ) (Figure ). .. The lambda DNA therefore contains methylated 5mC in the CpG context and unmethylated non-CpG, which is used to measure the efficiency of the bisulfite-mediated conversion and Tet oxidation.

    Article Title: Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974 ▿
    Article Snippet: Genetic complementation was used to test the in vivo activities of SerRS1 and SerRS2. .. The host strain was an E. coli mutant (K28) that is ineffective at making its own serS gene product at high temperature ( , ). serS1 and serS2 were separately cloned into a high-copy vector, pUC19, under the control of a constitutive glnS ′ promoter, which is a glnS mutant promoter used for aaRS expression in E. coli , and the resulting plasmids were transformed into E. coli K28. ..

    Article Title: CRISPR-Cas9-Guided Genome Engineering in C. elegans
    Article Snippet: Cas9 Expression Plasmid (Addgene plasmid #46168). pHKMC1 - Empty sgRNA Cloning Plasmid (Addgene plasmid #67720). pCFJ90 - P myo-2::mCherry::unc-54utr (Addgene plasmid #19327). pCFJ104 - P myo-3::mCherry::unc-54 (Addgene plasmid #19328). pMA122 - peel-1 negative selection (Addgene plasmid #34873, Optional). .. P eft-3 :: Cas9-SV40_NLS :: tbb-2 3’UTR (Addgene plasmid #46168). pUC19 (NEB N3041S). pPV477 (Addgene plasmid #42930). .. P myo-2::GFP ).

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
    Article Snippet: Genomic DNA concentrations were determined using the Qubit® 2.0 fluorometer (Invitrogen) and the quality of DNA was assessed by agarose gel electrophoresis. .. Preparation of 304 bp model DNA with 4mC modifications For N 4 -methylcytosine (4mC) containing model DNA, 0.5 ng of pUC19 vector DNA (NEB) was PCR amplified as follows in a 50 μl reaction: 2.5 U RedTaq polymerase (Sigma), 5 μl 10× reaction buffer, 1 μl N 4 -methyl-dCTP (4mdCTP) (Trilink)/dATP/dGTP/dTTP cocktail (10 mM each), 1 μl 10 mM forward primer (5′-GAACGAAAACTCACGTTAAGGG), 1 μl 10 mM reverse primer (5′-TGCTGATAAATCTGGAGCCG). ..

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: The resulting gel was stained with GelRed (Thermo Fisher Scientific Inc., Waltham, MA, USA). .. For cloning and transformation, both the PCR products and the pUC19 vector (New England Biolabs) were digested using HindIII-HF and XhoI restriction enzymes at 37 °C for 1 h in 1x CutSmart buffer (New England Biolabs). .. The digested products were then cleansed of extraneous DNA using the MinElute Reaction Cleanup Kit (QIAGEN, Germantown, MD).

    Generated:

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
    Article Snippet: .. The 4mC control is generated by PCR amplification with 4mdCTP using part of the pUC19 vector (∼1 kb) as a template, whereas the C/5mC control is generated by treating unmethylated lambda DNA with the CpG methyltransferase (M. SssI ) (Figure ). .. The lambda DNA therefore contains methylated 5mC in the CpG context and unmethylated non-CpG, which is used to measure the efficiency of the bisulfite-mediated conversion and Tet oxidation.

    Polymerase Chain Reaction:

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
    Article Snippet: .. The 4mC control is generated by PCR amplification with 4mdCTP using part of the pUC19 vector (∼1 kb) as a template, whereas the C/5mC control is generated by treating unmethylated lambda DNA with the CpG methyltransferase (M. SssI ) (Figure ). .. The lambda DNA therefore contains methylated 5mC in the CpG context and unmethylated non-CpG, which is used to measure the efficiency of the bisulfite-mediated conversion and Tet oxidation.

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
    Article Snippet: Genomic DNA concentrations were determined using the Qubit® 2.0 fluorometer (Invitrogen) and the quality of DNA was assessed by agarose gel electrophoresis. .. Preparation of 304 bp model DNA with 4mC modifications For N 4 -methylcytosine (4mC) containing model DNA, 0.5 ng of pUC19 vector DNA (NEB) was PCR amplified as follows in a 50 μl reaction: 2.5 U RedTaq polymerase (Sigma), 5 μl 10× reaction buffer, 1 μl N 4 -methyl-dCTP (4mdCTP) (Trilink)/dATP/dGTP/dTTP cocktail (10 mM each), 1 μl 10 mM forward primer (5′-GAACGAAAACTCACGTTAAGGG), 1 μl 10 mM reverse primer (5′-TGCTGATAAATCTGGAGCCG). ..

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: The resulting gel was stained with GelRed (Thermo Fisher Scientific Inc., Waltham, MA, USA). .. For cloning and transformation, both the PCR products and the pUC19 vector (New England Biolabs) were digested using HindIII-HF and XhoI restriction enzymes at 37 °C for 1 h in 1x CutSmart buffer (New England Biolabs). .. The digested products were then cleansed of extraneous DNA using the MinElute Reaction Cleanup Kit (QIAGEN, Germantown, MD).

    Amplification:

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
    Article Snippet: .. The 4mC control is generated by PCR amplification with 4mdCTP using part of the pUC19 vector (∼1 kb) as a template, whereas the C/5mC control is generated by treating unmethylated lambda DNA with the CpG methyltransferase (M. SssI ) (Figure ). .. The lambda DNA therefore contains methylated 5mC in the CpG context and unmethylated non-CpG, which is used to measure the efficiency of the bisulfite-mediated conversion and Tet oxidation.

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
    Article Snippet: Genomic DNA concentrations were determined using the Qubit® 2.0 fluorometer (Invitrogen) and the quality of DNA was assessed by agarose gel electrophoresis. .. Preparation of 304 bp model DNA with 4mC modifications For N 4 -methylcytosine (4mC) containing model DNA, 0.5 ng of pUC19 vector DNA (NEB) was PCR amplified as follows in a 50 μl reaction: 2.5 U RedTaq polymerase (Sigma), 5 μl 10× reaction buffer, 1 μl N 4 -methyl-dCTP (4mdCTP) (Trilink)/dATP/dGTP/dTTP cocktail (10 mM each), 1 μl 10 mM forward primer (5′-GAACGAAAACTCACGTTAAGGG), 1 μl 10 mM reverse primer (5′-TGCTGATAAATCTGGAGCCG). ..

    Lambda DNA Preparation:

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
    Article Snippet: .. The 4mC control is generated by PCR amplification with 4mdCTP using part of the pUC19 vector (∼1 kb) as a template, whereas the C/5mC control is generated by treating unmethylated lambda DNA with the CpG methyltransferase (M. SssI ) (Figure ). .. The lambda DNA therefore contains methylated 5mC in the CpG context and unmethylated non-CpG, which is used to measure the efficiency of the bisulfite-mediated conversion and Tet oxidation.

    Mutagenesis:

    Article Title: Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974 ▿
    Article Snippet: Genetic complementation was used to test the in vivo activities of SerRS1 and SerRS2. .. The host strain was an E. coli mutant (K28) that is ineffective at making its own serS gene product at high temperature ( , ). serS1 and serS2 were separately cloned into a high-copy vector, pUC19, under the control of a constitutive glnS ′ promoter, which is a glnS mutant promoter used for aaRS expression in E. coli , and the resulting plasmids were transformed into E. coli K28. ..

    Clone Assay:

    Article Title: Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974 ▿
    Article Snippet: Genetic complementation was used to test the in vivo activities of SerRS1 and SerRS2. .. The host strain was an E. coli mutant (K28) that is ineffective at making its own serS gene product at high temperature ( , ). serS1 and serS2 were separately cloned into a high-copy vector, pUC19, under the control of a constitutive glnS ′ promoter, which is a glnS mutant promoter used for aaRS expression in E. coli , and the resulting plasmids were transformed into E. coli K28. ..

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: The resulting gel was stained with GelRed (Thermo Fisher Scientific Inc., Waltham, MA, USA). .. For cloning and transformation, both the PCR products and the pUC19 vector (New England Biolabs) were digested using HindIII-HF and XhoI restriction enzymes at 37 °C for 1 h in 1x CutSmart buffer (New England Biolabs). .. The digested products were then cleansed of extraneous DNA using the MinElute Reaction Cleanup Kit (QIAGEN, Germantown, MD).

    Expressing:

    Article Title: Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974 ▿
    Article Snippet: Genetic complementation was used to test the in vivo activities of SerRS1 and SerRS2. .. The host strain was an E. coli mutant (K28) that is ineffective at making its own serS gene product at high temperature ( , ). serS1 and serS2 were separately cloned into a high-copy vector, pUC19, under the control of a constitutive glnS ′ promoter, which is a glnS mutant promoter used for aaRS expression in E. coli , and the resulting plasmids were transformed into E. coli K28. ..

    Transformation Assay:

    Article Title: Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974 ▿
    Article Snippet: Genetic complementation was used to test the in vivo activities of SerRS1 and SerRS2. .. The host strain was an E. coli mutant (K28) that is ineffective at making its own serS gene product at high temperature ( , ). serS1 and serS2 were separately cloned into a high-copy vector, pUC19, under the control of a constitutive glnS ′ promoter, which is a glnS mutant promoter used for aaRS expression in E. coli , and the resulting plasmids were transformed into E. coli K28. ..

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: The resulting gel was stained with GelRed (Thermo Fisher Scientific Inc., Waltham, MA, USA). .. For cloning and transformation, both the PCR products and the pUC19 vector (New England Biolabs) were digested using HindIII-HF and XhoI restriction enzymes at 37 °C for 1 h in 1x CutSmart buffer (New England Biolabs). .. The digested products were then cleansed of extraneous DNA using the MinElute Reaction Cleanup Kit (QIAGEN, Germantown, MD).

    Incubation:

    Article Title: C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents
    Article Snippet: .. Reactions were carried out according to the following general procedure: in a total volume of 20 μl using 80 mM HEPES buffer (pH 7.2) with 25 mM NaCl, 400 ng pUC19 (NEB, N3041) and varying concentrations of test compound (5, 10, 20 and 30 μM), samples were incubated at 37°C for both 5 and 12 h. Reactions were quenched by adding 6x loading buffer (Fermentas) containing 10 mM Tris-HCl, 0.03% bromophenol blue, 0.03% xylene cyanole FF, 60% glycerol, 60 mM EDTA and samples were loaded onto an agarose gel (1.2%) containing 3 μl EtBr. .. Electrophoresis was completed at 60 V for 1 h in 1x TAE buffer.

    Agarose Gel Electrophoresis:

    Article Title: C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents
    Article Snippet: .. Reactions were carried out according to the following general procedure: in a total volume of 20 μl using 80 mM HEPES buffer (pH 7.2) with 25 mM NaCl, 400 ng pUC19 (NEB, N3041) and varying concentrations of test compound (5, 10, 20 and 30 μM), samples were incubated at 37°C for both 5 and 12 h. Reactions were quenched by adding 6x loading buffer (Fermentas) containing 10 mM Tris-HCl, 0.03% bromophenol blue, 0.03% xylene cyanole FF, 60% glycerol, 60 mM EDTA and samples were loaded onto an agarose gel (1.2%) containing 3 μl EtBr. .. Electrophoresis was completed at 60 V for 1 h in 1x TAE buffer.

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    New England Biolabs puc19
    Cas9 variants have different cleavage activities against mismatched targets. ( A ) Representative agarose gels showing cleavage of a negatively supercoiled (nSC) plasmid containing the perfect target (0 MM) or mismatched (2 to 5 MM) target over a time course by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 15 s, 30 s, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h and 5 h. tr:crRNA = tracrRNA:crRNA. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (–) = pTarget or pLibrary alone incubated at 37°C for the longest time point in the assay (5 h); (-cr) = pTarget or pLibrary incubated with Cas9 only at 37°C for the longest time point in the assay (5 h); n = Nt.BspQI nicked <t>pUC19;</t> li = BsaI-HF linearized pUC19. ( B ) Quantification of supercoiled, linear and nicked pools from cleavage of perfect or fully crRNA-complementary (0 MM) and mismatched (2 to 5 MM) target plasmid by Cas9 after 10 min and 3 h. pTarget MM indicates target plasmid (0, 2 to 5 MM) alone incubated at 37°C for the time points indicated. Target sequences tested are listed with PAM (bold) and mismatches (lowercase and red) indicated. (–) indicates a cleavage reaction with the target plasmid and Cas9 only, and (+) indicates a cleavage reaction with the target plasmid, Cas9 and cognate tracrRNA:crRNA. Values plotted represent an average of three replicates. Error bars are SEM. * or • indicate P
    Puc19, supplied by New England Biolabs, 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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    New England Biolabs plasmid puc19
    Priming by g8 target variants. ( A ) Scheme of the E. coli KD263 CRISPR locus. The cas gene expression is controlled by inducible promoters. The CRISPR array consists of a single g8 spacer (blue boxes) surrounded by two repeats (black boxes). Priming is induced by transforming the cells with <t>pUC19</t> plasmids carrying the protospacer variants. Incorporation of new spacers (green box) is revealed using PCR amplification of the CRISPR array and agarose gel electrophoresis. ( B ) Incorporation of new spacers probed at different times after induction for the indicated g8 protospacer variants. ( C for other target variant plasmids). The height of the histogram bars corresponds to the number of HTS reads found for a particular position. The location of the priming protospacer and the PAM is shown as a blue-red box. The histogram entry in orange marks the hotspot HS1, which was used for semi-quantitative measurements of the primed adaptation efficiency (see E). ( D for correlation coefficients) is apparent. ( E ) Relative frequency of priming (i.e. CRISPR array extension) probed by qPCR with a primer specific for the frequently incorporated protospacer HS1 (see C) for the different target variants. Error bars represent the standard deviation of three repeat measurements.
    Plasmid Puc19, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cas9 variants have different cleavage activities against mismatched targets. ( A ) Representative agarose gels showing cleavage of a negatively supercoiled (nSC) plasmid containing the perfect target (0 MM) or mismatched (2 to 5 MM) target over a time course by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 15 s, 30 s, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h and 5 h. tr:crRNA = tracrRNA:crRNA. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (–) = pTarget or pLibrary alone incubated at 37°C for the longest time point in the assay (5 h); (-cr) = pTarget or pLibrary incubated with Cas9 only at 37°C for the longest time point in the assay (5 h); n = Nt.BspQI nicked pUC19; li = BsaI-HF linearized pUC19. ( B ) Quantification of supercoiled, linear and nicked pools from cleavage of perfect or fully crRNA-complementary (0 MM) and mismatched (2 to 5 MM) target plasmid by Cas9 after 10 min and 3 h. pTarget MM indicates target plasmid (0, 2 to 5 MM) alone incubated at 37°C for the time points indicated. Target sequences tested are listed with PAM (bold) and mismatches (lowercase and red) indicated. (–) indicates a cleavage reaction with the target plasmid and Cas9 only, and (+) indicates a cleavage reaction with the target plasmid, Cas9 and cognate tracrRNA:crRNA. Values plotted represent an average of three replicates. Error bars are SEM. * or • indicate P

    Journal: Nucleic Acids Research

    Article Title: Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR–Cas9 variants

    doi: 10.1093/nar/gkab163

    Figure Lengend Snippet: Cas9 variants have different cleavage activities against mismatched targets. ( A ) Representative agarose gels showing cleavage of a negatively supercoiled (nSC) plasmid containing the perfect target (0 MM) or mismatched (2 to 5 MM) target over a time course by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 15 s, 30 s, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h and 5 h. tr:crRNA = tracrRNA:crRNA. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (–) = pTarget or pLibrary alone incubated at 37°C for the longest time point in the assay (5 h); (-cr) = pTarget or pLibrary incubated with Cas9 only at 37°C for the longest time point in the assay (5 h); n = Nt.BspQI nicked pUC19; li = BsaI-HF linearized pUC19. ( B ) Quantification of supercoiled, linear and nicked pools from cleavage of perfect or fully crRNA-complementary (0 MM) and mismatched (2 to 5 MM) target plasmid by Cas9 after 10 min and 3 h. pTarget MM indicates target plasmid (0, 2 to 5 MM) alone incubated at 37°C for the time points indicated. Target sequences tested are listed with PAM (bold) and mismatches (lowercase and red) indicated. (–) indicates a cleavage reaction with the target plasmid and Cas9 only, and (+) indicates a cleavage reaction with the target plasmid, Cas9 and cognate tracrRNA:crRNA. Values plotted represent an average of three replicates. Error bars are SEM. * or • indicate P

    Article Snippet: For controls, pUC19 was prepared by restriction enzyme digestion using BsaI-HF to linearize the plasmid and Nt.BspQI to nick the plasmid using the manufacturer's protocols (New England Biolabs).

    Techniques: Plasmid Preparation, Incubation

    Priming by g8 target variants. ( A ) Scheme of the E. coli KD263 CRISPR locus. The cas gene expression is controlled by inducible promoters. The CRISPR array consists of a single g8 spacer (blue boxes) surrounded by two repeats (black boxes). Priming is induced by transforming the cells with pUC19 plasmids carrying the protospacer variants. Incorporation of new spacers (green box) is revealed using PCR amplification of the CRISPR array and agarose gel electrophoresis. ( B ) Incorporation of new spacers probed at different times after induction for the indicated g8 protospacer variants. ( C for other target variant plasmids). The height of the histogram bars corresponds to the number of HTS reads found for a particular position. The location of the priming protospacer and the PAM is shown as a blue-red box. The histogram entry in orange marks the hotspot HS1, which was used for semi-quantitative measurements of the primed adaptation efficiency (see E). ( D for correlation coefficients) is apparent. ( E ) Relative frequency of priming (i.e. CRISPR array extension) probed by qPCR with a primer specific for the frequently incorporated protospacer HS1 (see C) for the different target variants. Error bars represent the standard deviation of three repeat measurements.

    Journal: Nucleic Acids Research

    Article Title: Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro

    doi: 10.1093/nar/gky219

    Figure Lengend Snippet: Priming by g8 target variants. ( A ) Scheme of the E. coli KD263 CRISPR locus. The cas gene expression is controlled by inducible promoters. The CRISPR array consists of a single g8 spacer (blue boxes) surrounded by two repeats (black boxes). Priming is induced by transforming the cells with pUC19 plasmids carrying the protospacer variants. Incorporation of new spacers (green box) is revealed using PCR amplification of the CRISPR array and agarose gel electrophoresis. ( B ) Incorporation of new spacers probed at different times after induction for the indicated g8 protospacer variants. ( C for other target variant plasmids). The height of the histogram bars corresponds to the number of HTS reads found for a particular position. The location of the priming protospacer and the PAM is shown as a blue-red box. The histogram entry in orange marks the hotspot HS1, which was used for semi-quantitative measurements of the primed adaptation efficiency (see E). ( D for correlation coefficients) is apparent. ( E ) Relative frequency of priming (i.e. CRISPR array extension) probed by qPCR with a primer specific for the frequently incorporated protospacer HS1 (see C) for the different target variants. Error bars represent the standard deviation of three repeat measurements.

    Article Snippet: Constructs containing the WT g8 protospacer and its variants were cloned into plasmid pUC19 (NEB) at the single SmaI (NEB) site by blunt end ligation.

    Techniques: CRISPR, Expressing, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Variant Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    Data analysis of spike-in controls from MethylC-seq and 4mC-TAB-seq in the context of C. kristjanssonii genomic DNA. ( A ) Composition of pUC19 DNA, lambda DNA, and C. kristjanssonii genomic DNA. ( B ) The percentage of detected as cytosine reads on 4mC sites in untreated and Tet-treated samples. ( C ) The detected as cytosine reads percentage on unmodified cytosine sites (non-CpG context) and 5mC sites (CpG context) in untreated and Tet-treated samples. ( D ) Quantification of 4mC and 5mC in C. kristjanssonii genomic DNA, determined by LC-MS/MS and deep-sequencing respectively. Error bars indicate mean ± SD, n = 4.

    Journal: Nucleic Acids Research

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing

    doi: 10.1093/nar/gkv738

    Figure Lengend Snippet: Data analysis of spike-in controls from MethylC-seq and 4mC-TAB-seq in the context of C. kristjanssonii genomic DNA. ( A ) Composition of pUC19 DNA, lambda DNA, and C. kristjanssonii genomic DNA. ( B ) The percentage of detected as cytosine reads on 4mC sites in untreated and Tet-treated samples. ( C ) The detected as cytosine reads percentage on unmodified cytosine sites (non-CpG context) and 5mC sites (CpG context) in untreated and Tet-treated samples. ( D ) Quantification of 4mC and 5mC in C. kristjanssonii genomic DNA, determined by LC-MS/MS and deep-sequencing respectively. Error bars indicate mean ± SD, n = 4.

    Article Snippet: Preparation of 304 bp model DNA with 4mC modifications For N 4 -methylcytosine (4mC) containing model DNA, 0.5 ng of pUC19 vector DNA (NEB) was PCR amplified as follows in a 50 μl reaction: 2.5 U RedTaq polymerase (Sigma), 5 μl 10× reaction buffer, 1 μl N 4 -methyl-dCTP (4mdCTP) (Trilink)/dATP/dGTP/dTTP cocktail (10 mM each), 1 μl 10 mM forward primer (5′-GAACGAAAACTCACGTTAAGGG), 1 μl 10 mM reverse primer (5′-TGCTGATAAATCTGGAGCCG).

    Techniques: Lambda DNA Preparation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing

    EM-seq accurately represents methylation EM-seq and bisulfite libraries were made using 10, 50 and 200 ng of NA12878 DNA with control DNA (2 ng unmethylated lambda and 0.1 ng CpG methylated pUC19). Libraries were sequenced on an Illumina NovaSeq 6000 (2 x 100 bases). 324 million paired reads for each library were aligned to a human + control reference genome (see supplemental materials) using bwa-meth 0.2.2. and methylation information was extracted from the alignments using MethylDackel. The top and bottom strand CpGs were counted independently, yielding a maximum of 56 million possible CpG sites. (A) NA12878 EM-seq and whole genome bisulfite library (WGBS) methylation in CpG, CHH and CHG contexts are similarly represented. Methylation state for unmethylated lambda control and CpG methylated pUC19 control DNAs are shown in Supplemental Figure 5. (B) The number of CpGs covered for EM-seq and bisulfite libraries were calculated and graphed at minimum coverage depths of 1x through 21x. (C) The number of CpGs detected were compared between EM-seq and bisulfite libraries at 1x and 8x coverage depths. CpGs unique to EM-seq libraries, bisulfite libraries or those that were common to both are represented in the Venn diagrams. (D, E) Methylkit analysis at minimum 1x coverage shows good CpG methylation correlation between 10 ng and 200 ng NA12878 EM-seq libraries (D) and WGBS libraries (E). Methylation level correlations between inputs and replicates of EM-seq libraries are better than for WGBS libraries. The reduction in observations of disagreement (upper left and lower right corners) is particularly striking. Correlation between EM-seq and WGBS libraries at 10 ng, 50 ng, and 200 ng NA12878 DNA input are shown in Supplemental Figure 7.

    Journal: bioRxiv

    Article Title: EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA

    doi: 10.1101/2019.12.20.884692

    Figure Lengend Snippet: EM-seq accurately represents methylation EM-seq and bisulfite libraries were made using 10, 50 and 200 ng of NA12878 DNA with control DNA (2 ng unmethylated lambda and 0.1 ng CpG methylated pUC19). Libraries were sequenced on an Illumina NovaSeq 6000 (2 x 100 bases). 324 million paired reads for each library were aligned to a human + control reference genome (see supplemental materials) using bwa-meth 0.2.2. and methylation information was extracted from the alignments using MethylDackel. The top and bottom strand CpGs were counted independently, yielding a maximum of 56 million possible CpG sites. (A) NA12878 EM-seq and whole genome bisulfite library (WGBS) methylation in CpG, CHH and CHG contexts are similarly represented. Methylation state for unmethylated lambda control and CpG methylated pUC19 control DNAs are shown in Supplemental Figure 5. (B) The number of CpGs covered for EM-seq and bisulfite libraries were calculated and graphed at minimum coverage depths of 1x through 21x. (C) The number of CpGs detected were compared between EM-seq and bisulfite libraries at 1x and 8x coverage depths. CpGs unique to EM-seq libraries, bisulfite libraries or those that were common to both are represented in the Venn diagrams. (D, E) Methylkit analysis at minimum 1x coverage shows good CpG methylation correlation between 10 ng and 200 ng NA12878 EM-seq libraries (D) and WGBS libraries (E). Methylation level correlations between inputs and replicates of EM-seq libraries are better than for WGBS libraries. The reduction in observations of disagreement (upper left and lower right corners) is particularly striking. Correlation between EM-seq and WGBS libraries at 10 ng, 50 ng, and 200 ng NA12878 DNA input are shown in Supplemental Figure 7.

    Article Snippet: CpG methylated pUC19, was made by transforming pUC19 plasmid (NEB, Ipswich, MA) into dam-/dcm-competent E.coli cells (NEB, Ipswich, MA).

    Techniques: Methylation, CpG Methylation Assay