puc19 vector  (New England Biolabs)


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    Name:
    pUC19 Vector
    Description:
    pUC19 Vector 250 ug
    Catalog Number:
    n3041l
    Price:
    300
    Size:
    250 ug
    Category:
    Vectors Plasmids
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    Structured Review

    New England Biolabs puc19 vector
    pUC19 Vector
    pUC19 Vector 250 ug
    https://www.bioz.com/result/puc19 vector/product/New England Biolabs
    Average 99 stars, based on 347 article reviews
    Price from $9.99 to $1999.99
    puc19 vector - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) 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

    2) 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

    3) Product Images from "DNA oxidation profiles of copper phenanthrene chemical nucleases"

    Article Title: DNA oxidation profiles of copper phenanthrene chemical nucleases

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2015.00028

    Lane 1–4 (A–D) DNA cleavage reactions with 250 nM, 500 nM, 1.0 μM, and 2.5 μM test complex (A) Cu-Phen, (B) Cu-DPQ-Phen, (C) Cu-DPPZ-Phen, and (D) Cu-Terph, 400 ng superhelical pUC19 and 1 mM added Na-L-ascorbate incubated at 37°C for 30 min . Lanes 5–16 (A–D) DNA cleavage reactions in the presence of recognition elements, methyl green (MG), netropsin (Net), and [Co(NH 3 ) 6 ]Cl 3 (Co(III)), where 400 ng pUC19 was initially pre-treated with 8 μM of respective non-covalent binding control at 37° C for 45 min and then with 250 nM, 500 nM, 1 μM, and 2.5 μM test complex in the presence of 1 mM added Na-L-ascorbate at 37°C for 30 min.
    Figure Legend Snippet: Lane 1–4 (A–D) DNA cleavage reactions with 250 nM, 500 nM, 1.0 μM, and 2.5 μM test complex (A) Cu-Phen, (B) Cu-DPQ-Phen, (C) Cu-DPPZ-Phen, and (D) Cu-Terph, 400 ng superhelical pUC19 and 1 mM added Na-L-ascorbate incubated at 37°C for 30 min . Lanes 5–16 (A–D) DNA cleavage reactions in the presence of recognition elements, methyl green (MG), netropsin (Net), and [Co(NH 3 ) 6 ]Cl 3 (Co(III)), where 400 ng pUC19 was initially pre-treated with 8 μM of respective non-covalent binding control at 37° C for 45 min and then with 250 nM, 500 nM, 1 μM, and 2.5 μM test complex in the presence of 1 mM added Na-L-ascorbate at 37°C for 30 min.

    Techniques Used: Incubation, Binding Assay

    4) Product Images from "Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv738

    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.
    Figure Legend 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.

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

    5) Product Images from "Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv738

    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.
    Figure Legend 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.

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

    6) Product Images from "Depurination of colibactin-derived interstrand cross-links"

    Article Title: Depurination of colibactin-derived interstrand cross-links

    Journal: bioRxiv

    doi: 10.1101/869313

    DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by denaturing electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, reaction proceed for 1 h, 2 h, 4 h, 6 h, and 9 h. The DNA was isolated, purified, and analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 2 h).
    Figure Legend Snippet: DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by denaturing electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, reaction proceed for 1 h, 2 h, 4 h, 6 h, and 9 h. The DNA was isolated, purified, and analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 2 h).

    Techniques Used: Plasmid Preparation, Cleavage Assay, Electrophoresis, Mutagenesis, Isolation, Purification, Agarose Gel Electrophoresis

    Exposure of pUC19 plasmid DNA to clb + , followed by incubation with Endo IV leads to consumption of undamaged plasmid and formation of nicked and linearized DNA. This is not observed in the clb − or clbL mutant controls. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #4), reacted with buffer (Lane #5), reacted with Endonuclease IV (Lane #6); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #7), reacted with buffer (Lane #8), reacted with Endonuclease IV (Lane #9); circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli (Lane #10), reacted with buffer (Lane #11), reacted with Endonuclease IV (Lane #12). Conditions (Lane #4–#6): circular pUC19 DNA from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane # 4); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #5); the DNA (3.9 µM base pair) was further reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #6). Conditions (Lane #7–#9): circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli. in in M9-CA media for 4 h at 37 °C (Lane #7); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #8); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #9). Conditions (Lane #10–#12): circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli. in M9-CA media for 4 h at 37 °C (Lane #10); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #11); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #12). The DNA was not re-purified and was directly analyzed by native agarose gel electrophoresis (90 V, 1.5 hr).
    Figure Legend Snippet: Exposure of pUC19 plasmid DNA to clb + , followed by incubation with Endo IV leads to consumption of undamaged plasmid and formation of nicked and linearized DNA. This is not observed in the clb − or clbL mutant controls. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #4), reacted with buffer (Lane #5), reacted with Endonuclease IV (Lane #6); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #7), reacted with buffer (Lane #8), reacted with Endonuclease IV (Lane #9); circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli (Lane #10), reacted with buffer (Lane #11), reacted with Endonuclease IV (Lane #12). Conditions (Lane #4–#6): circular pUC19 DNA from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane # 4); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #5); the DNA (3.9 µM base pair) was further reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #6). Conditions (Lane #7–#9): circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli. in in M9-CA media for 4 h at 37 °C (Lane #7); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #8); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #9). Conditions (Lane #10–#12): circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli. in M9-CA media for 4 h at 37 °C (Lane #10); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #11); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #12). The DNA was not re-purified and was directly analyzed by native agarose gel electrophoresis (90 V, 1.5 hr).

    Techniques Used: Plasmid Preparation, Incubation, Mutagenesis, Isolation, Co-Culture Assay, Purification, Agarose Gel Electrophoresis

    Formation of DNA SSBs and DSBs in circular pUC19 plasmid DNA treated with clb + or clb − E. coli . A. SSBs as a function of time. B. DSBs as a function of time. SSBs and DSBs are expressed as intensity of their respective bands relative to an internal control (untreated DNA). Three technical replicates (Fig. S2).
    Figure Legend Snippet: Formation of DNA SSBs and DSBs in circular pUC19 plasmid DNA treated with clb + or clb − E. coli . A. SSBs as a function of time. B. DSBs as a function of time. SSBs and DSBs are expressed as intensity of their respective bands relative to an internal control (untreated DNA). Three technical replicates (Fig. S2).

    Techniques Used: Plasmid Preparation

    Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).
    Figure Legend Snippet: Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).

    Techniques Used: Isolation, Co-Culture Assay, Positive Control, Nucleic Acid Electrophoresis, Cell Culture, Agarose Gel Electrophoresis

    Analysis of induction of AP sites by the colibactin precursor 4. A. Incubation of plasmid pUC19 DNA exposed to 4 in buffer for 18 h results in minor nicking and cleavage. B. Addition of EndoIV increases the amount of nicked and cleaved plasmid. Conditions: A. 5% DMSO was used as vehicle (negative control), and 100 µM cisplatin was used as positive control. DNA ladder (Lane #1); linearized pUC19 DNA standard (Lane #2); 5% DMSO (Lane #3); 100 µM cisplatin (Lane #4); 100 µM 4 (Lane #5); 10 µM 4 (Lane #6); 1 µM 4 (Lane #7); 100 nM 4 (Lane #8); 10 nM 4 (Lane #9). Conditions (Lane #3): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lane #4): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 100 µM cisplatin, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #5–#9): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–10 nM), 5% DMSO, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. The DNA was analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 1.5 h). B. 5% DMSO was used as vehicle. DNA ladder (Lane #1); 5% DMSO (Lane #2); 100 µM 4 (Lane #3); 10 µM 4 (Lane #4); 1 µM 4 (Lane #5); post buffer-reacted after 10 µM 4 (Lane #6); post buffer-reacted after 1 µM 4 (Lane #7); post EndoIV-reacted after 10 µM 4 (Lane #8); post EndoIV-reacted after 1 µM 4 (Lane #9); circular pUC19 plasmid standard (Lane #10); linearized pUC19 plasmid standard (Lane #11). Conditions (Lane #2): circular pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #3–#5): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–1 µM), 5% DMSO, 10 mM citric buffer, pH 5.0, 3 h, 37 °C. Conditions (Lanes #6–#7): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. Conditions (Lanes #8–#9): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), 20 units of Endonuclease IV (New England Biolabs®), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. The DNA was analyzed by native agarose gel electrophoresis (90 V, 2 h).
    Figure Legend Snippet: Analysis of induction of AP sites by the colibactin precursor 4. A. Incubation of plasmid pUC19 DNA exposed to 4 in buffer for 18 h results in minor nicking and cleavage. B. Addition of EndoIV increases the amount of nicked and cleaved plasmid. Conditions: A. 5% DMSO was used as vehicle (negative control), and 100 µM cisplatin was used as positive control. DNA ladder (Lane #1); linearized pUC19 DNA standard (Lane #2); 5% DMSO (Lane #3); 100 µM cisplatin (Lane #4); 100 µM 4 (Lane #5); 10 µM 4 (Lane #6); 1 µM 4 (Lane #7); 100 nM 4 (Lane #8); 10 nM 4 (Lane #9). Conditions (Lane #3): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lane #4): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 100 µM cisplatin, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #5–#9): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–10 nM), 5% DMSO, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. The DNA was analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 1.5 h). B. 5% DMSO was used as vehicle. DNA ladder (Lane #1); 5% DMSO (Lane #2); 100 µM 4 (Lane #3); 10 µM 4 (Lane #4); 1 µM 4 (Lane #5); post buffer-reacted after 10 µM 4 (Lane #6); post buffer-reacted after 1 µM 4 (Lane #7); post EndoIV-reacted after 10 µM 4 (Lane #8); post EndoIV-reacted after 1 µM 4 (Lane #9); circular pUC19 plasmid standard (Lane #10); linearized pUC19 plasmid standard (Lane #11). Conditions (Lane #2): circular pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #3–#5): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–1 µM), 5% DMSO, 10 mM citric buffer, pH 5.0, 3 h, 37 °C. Conditions (Lanes #6–#7): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. Conditions (Lanes #8–#9): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), 20 units of Endonuclease IV (New England Biolabs®), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. The DNA was analyzed by native agarose gel electrophoresis (90 V, 2 h).

    Techniques Used: Incubation, Plasmid Preparation, Negative Control, Positive Control, Agarose Gel Electrophoresis

    DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by neutral gel electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, 1 h, 2 h, 4 h, 6 h, or 9 h. The DNA was isolated, purified, and analyzed by native agarose gel electrophoresis (90 V, 2 h). Order of elution (bottom to top): supercoiled plasmid DNA, linearized DNA, nicked DNA.
    Figure Legend Snippet: DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by neutral gel electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, 1 h, 2 h, 4 h, 6 h, or 9 h. The DNA was isolated, purified, and analyzed by native agarose gel electrophoresis (90 V, 2 h). Order of elution (bottom to top): supercoiled plasmid DNA, linearized DNA, nicked DNA.

    Techniques Used: Plasmid Preparation, Cleavage Assay, Nucleic Acid Electrophoresis, Mutagenesis, Isolation, Purification, Agarose Gel Electrophoresis

    7) Product Images from "Synthesis of the unnatural amino acid Nα-Nε-(ferrocene-1-acetyl)-l-lysine: a novel organometallic nuclease"

    Article Title: Synthesis of the unnatural amino acid Nα-Nε-(ferrocene-1-acetyl)-l-lysine: a novel organometallic nuclease

    Journal: Journal of organometallic chemistry

    doi: 10.1016/j.jorganchem.2008.06.012

    DNA cleavage assays. A 20 μl reaction containing pUC19 DNA (1.0 μg, 75 μM bp) was incubated with 0-150 μM of 1 (a) or ferrocene acetic acid (b) at 25 °C for 16 h. Separation of supercoiled (SC), nicked circular (NC), and linear (L) forms of pUC19 DNA in each reaction was accomplished by agarose (1%) gel electrophoresis.
    Figure Legend Snippet: DNA cleavage assays. A 20 μl reaction containing pUC19 DNA (1.0 μg, 75 μM bp) was incubated with 0-150 μM of 1 (a) or ferrocene acetic acid (b) at 25 °C for 16 h. Separation of supercoiled (SC), nicked circular (NC), and linear (L) forms of pUC19 DNA in each reaction was accomplished by agarose (1%) gel electrophoresis.

    Techniques Used: Incubation, Nucleic Acid Electrophoresis

    8) Product Images from "Depurination of colibactin-derived interstrand cross-links"

    Article Title: Depurination of colibactin-derived interstrand cross-links

    Journal: bioRxiv

    doi: 10.1101/869313

    DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by denaturing electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, reaction proceed for 1 h, 2 h, 4 h, 6 h, and 9 h. The DNA was isolated, purified, and analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 2 h).
    Figure Legend Snippet: DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by denaturing electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, reaction proceed for 1 h, 2 h, 4 h, 6 h, and 9 h. The DNA was isolated, purified, and analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 2 h).

    Techniques Used: Plasmid Preparation, Cleavage Assay, Electrophoresis, Mutagenesis, Isolation, Purification, Agarose Gel Electrophoresis

    Exposure of pUC19 plasmid DNA to clb + , followed by incubation with Endo IV leads to consumption of undamaged plasmid and formation of nicked and linearized DNA. This is not observed in the clb − or clbL mutant controls. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #4), reacted with buffer (Lane #5), reacted with Endonuclease IV (Lane #6); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #7), reacted with buffer (Lane #8), reacted with Endonuclease IV (Lane #9); circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli (Lane #10), reacted with buffer (Lane #11), reacted with Endonuclease IV (Lane #12). Conditions (Lane #4–#6): circular pUC19 DNA from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane # 4); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #5); the DNA (3.9 µM base pair) was further reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #6). Conditions (Lane #7–#9): circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli. in in M9-CA media for 4 h at 37 °C (Lane #7); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #8); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #9). Conditions (Lane #10–#12): circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli. in M9-CA media for 4 h at 37 °C (Lane #10); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #11); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #12). The DNA was not re-purified and was directly analyzed by native agarose gel electrophoresis (90 V, 1.5 hr).
    Figure Legend Snippet: Exposure of pUC19 plasmid DNA to clb + , followed by incubation with Endo IV leads to consumption of undamaged plasmid and formation of nicked and linearized DNA. This is not observed in the clb − or clbL mutant controls. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #4), reacted with buffer (Lane #5), reacted with Endonuclease IV (Lane #6); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #7), reacted with buffer (Lane #8), reacted with Endonuclease IV (Lane #9); circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli (Lane #10), reacted with buffer (Lane #11), reacted with Endonuclease IV (Lane #12). Conditions (Lane #4–#6): circular pUC19 DNA from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane # 4); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #5); the DNA (3.9 µM base pair) was further reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #6). Conditions (Lane #7–#9): circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli. in in M9-CA media for 4 h at 37 °C (Lane #7); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #8); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #9). Conditions (Lane #10–#12): circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli. in M9-CA media for 4 h at 37 °C (Lane #10); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #11); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #12). The DNA was not re-purified and was directly analyzed by native agarose gel electrophoresis (90 V, 1.5 hr).

    Techniques Used: Plasmid Preparation, Incubation, Mutagenesis, Isolation, Co-Culture Assay, Purification, Agarose Gel Electrophoresis

    Formation of DNA SSBs and DSBs in circular pUC19 plasmid DNA treated with clb + or clb − E. coli . A. SSBs as a function of time. B. DSBs as a function of time. SSBs and DSBs are expressed as intensity of their respective bands relative to an internal control (untreated DNA). Three technical replicates (Fig. S2).
    Figure Legend Snippet: Formation of DNA SSBs and DSBs in circular pUC19 plasmid DNA treated with clb + or clb − E. coli . A. SSBs as a function of time. B. DSBs as a function of time. SSBs and DSBs are expressed as intensity of their respective bands relative to an internal control (untreated DNA). Three technical replicates (Fig. S2).

    Techniques Used: Plasmid Preparation

    Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).
    Figure Legend Snippet: Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).

    Techniques Used: Isolation, Co-Culture Assay, Positive Control, Nucleic Acid Electrophoresis, Cell Culture, Agarose Gel Electrophoresis

    Analysis of induction of AP sites by the colibactin precursor 4. A. Incubation of plasmid pUC19 DNA exposed to 4 in buffer for 18 h results in minor nicking and cleavage. B. Addition of EndoIV increases the amount of nicked and cleaved plasmid. Conditions: A. 5% DMSO was used as vehicle (negative control), and 100 µM cisplatin was used as positive control. DNA ladder (Lane #1); linearized pUC19 DNA standard (Lane #2); 5% DMSO (Lane #3); 100 µM cisplatin (Lane #4); 100 µM 4 (Lane #5); 10 µM 4 (Lane #6); 1 µM 4 (Lane #7); 100 nM 4 (Lane #8); 10 nM 4 (Lane #9). Conditions (Lane #3): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lane #4): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 100 µM cisplatin, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #5–#9): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–10 nM), 5% DMSO, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. The DNA was analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 1.5 h). B. 5% DMSO was used as vehicle. DNA ladder (Lane #1); 5% DMSO (Lane #2); 100 µM 4 (Lane #3); 10 µM 4 (Lane #4); 1 µM 4 (Lane #5); post buffer-reacted after 10 µM 4 (Lane #6); post buffer-reacted after 1 µM 4 (Lane #7); post EndoIV-reacted after 10 µM 4 (Lane #8); post EndoIV-reacted after 1 µM 4 (Lane #9); circular pUC19 plasmid standard (Lane #10); linearized pUC19 plasmid standard (Lane #11). Conditions (Lane #2): circular pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #3–#5): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–1 µM), 5% DMSO, 10 mM citric buffer, pH 5.0, 3 h, 37 °C. Conditions (Lanes #6–#7): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. Conditions (Lanes #8–#9): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), 20 units of Endonuclease IV (New England Biolabs®), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. The DNA was analyzed by native agarose gel electrophoresis (90 V, 2 h).
    Figure Legend Snippet: Analysis of induction of AP sites by the colibactin precursor 4. A. Incubation of plasmid pUC19 DNA exposed to 4 in buffer for 18 h results in minor nicking and cleavage. B. Addition of EndoIV increases the amount of nicked and cleaved plasmid. Conditions: A. 5% DMSO was used as vehicle (negative control), and 100 µM cisplatin was used as positive control. DNA ladder (Lane #1); linearized pUC19 DNA standard (Lane #2); 5% DMSO (Lane #3); 100 µM cisplatin (Lane #4); 100 µM 4 (Lane #5); 10 µM 4 (Lane #6); 1 µM 4 (Lane #7); 100 nM 4 (Lane #8); 10 nM 4 (Lane #9). Conditions (Lane #3): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lane #4): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 100 µM cisplatin, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #5–#9): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–10 nM), 5% DMSO, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. The DNA was analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 1.5 h). B. 5% DMSO was used as vehicle. DNA ladder (Lane #1); 5% DMSO (Lane #2); 100 µM 4 (Lane #3); 10 µM 4 (Lane #4); 1 µM 4 (Lane #5); post buffer-reacted after 10 µM 4 (Lane #6); post buffer-reacted after 1 µM 4 (Lane #7); post EndoIV-reacted after 10 µM 4 (Lane #8); post EndoIV-reacted after 1 µM 4 (Lane #9); circular pUC19 plasmid standard (Lane #10); linearized pUC19 plasmid standard (Lane #11). Conditions (Lane #2): circular pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #3–#5): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–1 µM), 5% DMSO, 10 mM citric buffer, pH 5.0, 3 h, 37 °C. Conditions (Lanes #6–#7): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. Conditions (Lanes #8–#9): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), 20 units of Endonuclease IV (New England Biolabs®), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. The DNA was analyzed by native agarose gel electrophoresis (90 V, 2 h).

    Techniques Used: Incubation, Plasmid Preparation, Negative Control, Positive Control, Agarose Gel Electrophoresis

    DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by neutral gel electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, 1 h, 2 h, 4 h, 6 h, or 9 h. The DNA was isolated, purified, and analyzed by native agarose gel electrophoresis (90 V, 2 h). Order of elution (bottom to top): supercoiled plasmid DNA, linearized DNA, nicked DNA.
    Figure Legend Snippet: DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by neutral gel electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, 1 h, 2 h, 4 h, 6 h, or 9 h. The DNA was isolated, purified, and analyzed by native agarose gel electrophoresis (90 V, 2 h). Order of elution (bottom to top): supercoiled plasmid DNA, linearized DNA, nicked DNA.

    Techniques Used: Plasmid Preparation, Cleavage Assay, Nucleic Acid Electrophoresis, Mutagenesis, Isolation, Purification, Agarose Gel Electrophoresis

    9) 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

    10) 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

    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

    12) Product Images from "Depurination of colibactin-derived interstrand cross-links"

    Article Title: Depurination of colibactin-derived interstrand cross-links

    Journal: bioRxiv

    doi: 10.1101/869313

    DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by denaturing electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, reaction proceed for 1 h, 2 h, 4 h, 6 h, and 9 h. The DNA was isolated, purified, and analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 2 h).
    Figure Legend Snippet: DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by denaturing electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, reaction proceed for 1 h, 2 h, 4 h, 6 h, and 9 h. The DNA was isolated, purified, and analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 2 h).

    Techniques Used: Plasmid Preparation, Cleavage Assay, Electrophoresis, Mutagenesis, Isolation, Purification, Agarose Gel Electrophoresis

    Exposure of pUC19 plasmid DNA to clb + , followed by incubation with Endo IV leads to consumption of undamaged plasmid and formation of nicked and linearized DNA. This is not observed in the clb − or clbL mutant controls. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #4), reacted with buffer (Lane #5), reacted with Endonuclease IV (Lane #6); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #7), reacted with buffer (Lane #8), reacted with Endonuclease IV (Lane #9); circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli (Lane #10), reacted with buffer (Lane #11), reacted with Endonuclease IV (Lane #12). Conditions (Lane #4–#6): circular pUC19 DNA from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane # 4); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #5); the DNA (3.9 µM base pair) was further reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #6). Conditions (Lane #7–#9): circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli. in in M9-CA media for 4 h at 37 °C (Lane #7); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #8); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #9). Conditions (Lane #10–#12): circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli. in M9-CA media for 4 h at 37 °C (Lane #10); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #11); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #12). The DNA was not re-purified and was directly analyzed by native agarose gel electrophoresis (90 V, 1.5 hr).
    Figure Legend Snippet: Exposure of pUC19 plasmid DNA to clb + , followed by incubation with Endo IV leads to consumption of undamaged plasmid and formation of nicked and linearized DNA. This is not observed in the clb − or clbL mutant controls. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #4), reacted with buffer (Lane #5), reacted with Endonuclease IV (Lane #6); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #7), reacted with buffer (Lane #8), reacted with Endonuclease IV (Lane #9); circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli (Lane #10), reacted with buffer (Lane #11), reacted with Endonuclease IV (Lane #12). Conditions (Lane #4–#6): circular pUC19 DNA from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane # 4); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #5); the DNA (3.9 µM base pair) was further reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #6). Conditions (Lane #7–#9): circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli. in in M9-CA media for 4 h at 37 °C (Lane #7); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #8); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #9). Conditions (Lane #10–#12): circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli. in M9-CA media for 4 h at 37 °C (Lane #10); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #11); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #12). The DNA was not re-purified and was directly analyzed by native agarose gel electrophoresis (90 V, 1.5 hr).

    Techniques Used: Plasmid Preparation, Incubation, Mutagenesis, Isolation, Co-Culture Assay, Purification, Agarose Gel Electrophoresis

    Formation of DNA SSBs and DSBs in circular pUC19 plasmid DNA treated with clb + or clb − E. coli . A. SSBs as a function of time. B. DSBs as a function of time. SSBs and DSBs are expressed as intensity of their respective bands relative to an internal control (untreated DNA). Three technical replicates (Fig. S2).
    Figure Legend Snippet: Formation of DNA SSBs and DSBs in circular pUC19 plasmid DNA treated with clb + or clb − E. coli . A. SSBs as a function of time. B. DSBs as a function of time. SSBs and DSBs are expressed as intensity of their respective bands relative to an internal control (untreated DNA). Three technical replicates (Fig. S2).

    Techniques Used: Plasmid Preparation

    Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).
    Figure Legend Snippet: Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).

    Techniques Used: Isolation, Co-Culture Assay, Positive Control, Nucleic Acid Electrophoresis, Cell Culture, Agarose Gel Electrophoresis

    Analysis of induction of AP sites by the colibactin precursor 4. A. Incubation of plasmid pUC19 DNA exposed to 4 in buffer for 18 h results in minor nicking and cleavage. B. Addition of EndoIV increases the amount of nicked and cleaved plasmid. Conditions: A. 5% DMSO was used as vehicle (negative control), and 100 µM cisplatin was used as positive control. DNA ladder (Lane #1); linearized pUC19 DNA standard (Lane #2); 5% DMSO (Lane #3); 100 µM cisplatin (Lane #4); 100 µM 4 (Lane #5); 10 µM 4 (Lane #6); 1 µM 4 (Lane #7); 100 nM 4 (Lane #8); 10 nM 4 (Lane #9). Conditions (Lane #3): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lane #4): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 100 µM cisplatin, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #5–#9): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–10 nM), 5% DMSO, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. The DNA was analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 1.5 h). B. 5% DMSO was used as vehicle. DNA ladder (Lane #1); 5% DMSO (Lane #2); 100 µM 4 (Lane #3); 10 µM 4 (Lane #4); 1 µM 4 (Lane #5); post buffer-reacted after 10 µM 4 (Lane #6); post buffer-reacted after 1 µM 4 (Lane #7); post EndoIV-reacted after 10 µM 4 (Lane #8); post EndoIV-reacted after 1 µM 4 (Lane #9); circular pUC19 plasmid standard (Lane #10); linearized pUC19 plasmid standard (Lane #11). Conditions (Lane #2): circular pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #3–#5): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–1 µM), 5% DMSO, 10 mM citric buffer, pH 5.0, 3 h, 37 °C. Conditions (Lanes #6–#7): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. Conditions (Lanes #8–#9): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), 20 units of Endonuclease IV (New England Biolabs®), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. The DNA was analyzed by native agarose gel electrophoresis (90 V, 2 h).
    Figure Legend Snippet: Analysis of induction of AP sites by the colibactin precursor 4. A. Incubation of plasmid pUC19 DNA exposed to 4 in buffer for 18 h results in minor nicking and cleavage. B. Addition of EndoIV increases the amount of nicked and cleaved plasmid. Conditions: A. 5% DMSO was used as vehicle (negative control), and 100 µM cisplatin was used as positive control. DNA ladder (Lane #1); linearized pUC19 DNA standard (Lane #2); 5% DMSO (Lane #3); 100 µM cisplatin (Lane #4); 100 µM 4 (Lane #5); 10 µM 4 (Lane #6); 1 µM 4 (Lane #7); 100 nM 4 (Lane #8); 10 nM 4 (Lane #9). Conditions (Lane #3): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lane #4): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 100 µM cisplatin, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #5–#9): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–10 nM), 5% DMSO, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. The DNA was analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 1.5 h). B. 5% DMSO was used as vehicle. DNA ladder (Lane #1); 5% DMSO (Lane #2); 100 µM 4 (Lane #3); 10 µM 4 (Lane #4); 1 µM 4 (Lane #5); post buffer-reacted after 10 µM 4 (Lane #6); post buffer-reacted after 1 µM 4 (Lane #7); post EndoIV-reacted after 10 µM 4 (Lane #8); post EndoIV-reacted after 1 µM 4 (Lane #9); circular pUC19 plasmid standard (Lane #10); linearized pUC19 plasmid standard (Lane #11). Conditions (Lane #2): circular pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #3–#5): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–1 µM), 5% DMSO, 10 mM citric buffer, pH 5.0, 3 h, 37 °C. Conditions (Lanes #6–#7): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. Conditions (Lanes #8–#9): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), 20 units of Endonuclease IV (New England Biolabs®), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. The DNA was analyzed by native agarose gel electrophoresis (90 V, 2 h).

    Techniques Used: Incubation, Plasmid Preparation, Negative Control, Positive Control, Agarose Gel Electrophoresis

    DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by neutral gel electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, 1 h, 2 h, 4 h, 6 h, or 9 h. The DNA was isolated, purified, and analyzed by native agarose gel electrophoresis (90 V, 2 h). Order of elution (bottom to top): supercoiled plasmid DNA, linearized DNA, nicked DNA.
    Figure Legend Snippet: DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by neutral gel electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, 1 h, 2 h, 4 h, 6 h, or 9 h. The DNA was isolated, purified, and analyzed by native agarose gel electrophoresis (90 V, 2 h). Order of elution (bottom to top): supercoiled plasmid DNA, linearized DNA, nicked DNA.

    Techniques Used: Plasmid Preparation, Cleavage Assay, Nucleic Acid Electrophoresis, Mutagenesis, Isolation, Purification, Agarose Gel Electrophoresis

    13) Product Images from "Ultra-Low Background DNA Cloning System"

    Article Title: Ultra-Low Background DNA Cloning System

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0056530

    Restriction analysis of plasmids from non- GFPuv -expressing colonies. Plasmids from 13 non- GFPuv -expressing colonies were digested with Eco RV and Bam HI. (A) Diagram showing the restriction patterns of pUC19 carrying the conversion cassette SU32 and the IS5-inserted GFPuv gene (colony 9). IS5: transposable element in E. coli . (B) Diagram showing the restriction pattern of undigested pUC19 carrying the conversion cassette SU32 (colony 10). (C) Digested plasmids were analyzed by electrophoresis on 1% agarose gel. GeneRuler 1-kb DNA ladder (Fermentas) is indicated on the left (M).
    Figure Legend Snippet: Restriction analysis of plasmids from non- GFPuv -expressing colonies. Plasmids from 13 non- GFPuv -expressing colonies were digested with Eco RV and Bam HI. (A) Diagram showing the restriction patterns of pUC19 carrying the conversion cassette SU32 and the IS5-inserted GFPuv gene (colony 9). IS5: transposable element in E. coli . (B) Diagram showing the restriction pattern of undigested pUC19 carrying the conversion cassette SU32 (colony 10). (C) Digested plasmids were analyzed by electrophoresis on 1% agarose gel. GeneRuler 1-kb DNA ladder (Fermentas) is indicated on the left (M).

    Techniques Used: Expressing, Electrophoresis, Agarose Gel Electrophoresis

    DNA sequences of the crossover regions of Eco RI/ Xba I-digested pUC19 and GFPuv . The cross represents homologous recombination. The representative restriction sites are shown.
    Figure Legend Snippet: DNA sequences of the crossover regions of Eco RI/ Xba I-digested pUC19 and GFPuv . The cross represents homologous recombination. The representative restriction sites are shown.

    Techniques Used: Homologous Recombination

    Restriction analysis of plasmids from colonies expressing GFPuv . Plasmids from 8 colonies expressing GFPuv were digested with Eco RV and Bam HI. (A) Diagram showing the restriction patterns of pUC19 carrying the conversion cassette SU32 and GFPuv . (B) Digested plasmids were analyzed by electrophoresis on 1% agarose gel. GeneRuler 1-kb DNA ladder (Fermentas) is indicated on the left (M).
    Figure Legend Snippet: Restriction analysis of plasmids from colonies expressing GFPuv . Plasmids from 8 colonies expressing GFPuv were digested with Eco RV and Bam HI. (A) Diagram showing the restriction patterns of pUC19 carrying the conversion cassette SU32 and GFPuv . (B) Digested plasmids were analyzed by electrophoresis on 1% agarose gel. GeneRuler 1-kb DNA ladder (Fermentas) is indicated on the left (M).

    Techniques Used: Expressing, Electrophoresis, Agarose Gel Electrophoresis

    14) Product Images from "Di-copper metallodrugs promote NCI-60 chemotherapy via singlet oxygen and superoxide production with tandem TA/TA and AT/AT oligonucleotide discrimination"

    Article Title: Di-copper metallodrugs promote NCI-60 chemotherapy via singlet oxygen and superoxide production with tandem TA/TA and AT/AT oligonucleotide discrimination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky105

    ( A ) Mean tail moments of untreated SKOV-3 cells and 1.0 μM Cu-Oda, Cu-Terph and Dox. ( B ) Topoisomerase I unwinding across concentration range 0.10–400 μM for Cu-Oda and Cu-Terph . ( C ). DSBs induced by Cu-Oda and Cu-Terph , detected by immunostaining of γH2AX with MFI presented for di -Cu 2+ complexes and Dox. ( D ) CD spectra of Cu-Oda with stDNA and alternating co-polymers poly[d(A⋅T) 2 ] and poly[d(G⋅C) 2 ] (100 μM) at loading ratios 0.01–0.075.
    Figure Legend Snippet: ( A ) Mean tail moments of untreated SKOV-3 cells and 1.0 μM Cu-Oda, Cu-Terph and Dox. ( B ) Topoisomerase I unwinding across concentration range 0.10–400 μM for Cu-Oda and Cu-Terph . ( C ). DSBs induced by Cu-Oda and Cu-Terph , detected by immunostaining of γH2AX with MFI presented for di -Cu 2+ complexes and Dox. ( D ) CD spectra of Cu-Oda with stDNA and alternating co-polymers poly[d(A⋅T) 2 ] and poly[d(G⋅C) 2 ] (100 μM) at loading ratios 0.01–0.075.

    Techniques Used: Concentration Assay, Immunostaining

    15) Product Images from "Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv738

    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.
    Figure Legend 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.

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

    16) Product Images from "DNA oxidation profiles of copper phenanthrene chemical nucleases"

    Article Title: DNA oxidation profiles of copper phenanthrene chemical nucleases

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2015.00028

    DNA cleavage reactions in the presence of ROS scavengers . 400 ng of SC pUC19 was incubated for for 30 min at 37°C with 250 nM, 500 nM, 1 μM, and 2.5 μM of test complex (A) Cu-Phen, (B) Cu-DPQ-Phen, (C) Cu-DPPZ-Phen, and (D) Cu-Terph in the presence of 1 mM added Na-L-ascorbate for 30 min. Lanes 1–4 metal complex only, lanes 5–8, complex + 10 mM NaN 3 ; lanes 9–12, complex + 10 mM KI; lanes 13–16, complex + 10% DMSO; and lanes 17–20, complex + 77% D 2 O.
    Figure Legend Snippet: DNA cleavage reactions in the presence of ROS scavengers . 400 ng of SC pUC19 was incubated for for 30 min at 37°C with 250 nM, 500 nM, 1 μM, and 2.5 μM of test complex (A) Cu-Phen, (B) Cu-DPQ-Phen, (C) Cu-DPPZ-Phen, and (D) Cu-Terph in the presence of 1 mM added Na-L-ascorbate for 30 min. Lanes 1–4 metal complex only, lanes 5–8, complex + 10 mM NaN 3 ; lanes 9–12, complex + 10 mM KI; lanes 13–16, complex + 10% DMSO; and lanes 17–20, complex + 77% D 2 O.

    Techniques Used: Incubation

    Structure and quantification of 8-oxo-dG . Graph represents level of generated 8-oxo-dG as nM (left axis) and ng/mL (right axis). 3000 ng of SC pUC19 with 10 and 20 μM of test complexes Cu-Phen, Cu-DPQ-Phen, Cu-DPPZ-Phen, and Cu-Terph with 1 mM Na-L-ascorbate were incubated at 37°C for 30 min and followed by ELISA protocol.
    Figure Legend Snippet: Structure and quantification of 8-oxo-dG . Graph represents level of generated 8-oxo-dG as nM (left axis) and ng/mL (right axis). 3000 ng of SC pUC19 with 10 and 20 μM of test complexes Cu-Phen, Cu-DPQ-Phen, Cu-DPPZ-Phen, and Cu-Terph with 1 mM Na-L-ascorbate were incubated at 37°C for 30 min and followed by ELISA protocol.

    Techniques Used: Generated, Incubation, Enzyme-linked Immunosorbent Assay

    Lane 1–4 (A–D) DNA cleavage reactions with 250 nM, 500 nM, 1.0 μM, and 2.5 μM test complex (A) Cu-Phen, (B) Cu-DPQ-Phen, (C) Cu-DPPZ-Phen, and (D) Cu-Terph, 400 ng superhelical pUC19 and 1 mM added Na-L-ascorbate incubated at 37°C for 30 min . Lanes 5–16 (A–D) DNA cleavage reactions in the presence of recognition elements, methyl green (MG), netropsin (Net), and [Co(NH 3 ) 6 ]Cl 3 (Co(III)), where 400 ng pUC19 was initially pre-treated with 8 μM of respective non-covalent binding control at 37° C for 45 min and then with 250 nM, 500 nM, 1 μM, and 2.5 μM test complex in the presence of 1 mM added Na-L-ascorbate at 37°C for 30 min.
    Figure Legend Snippet: Lane 1–4 (A–D) DNA cleavage reactions with 250 nM, 500 nM, 1.0 μM, and 2.5 μM test complex (A) Cu-Phen, (B) Cu-DPQ-Phen, (C) Cu-DPPZ-Phen, and (D) Cu-Terph, 400 ng superhelical pUC19 and 1 mM added Na-L-ascorbate incubated at 37°C for 30 min . Lanes 5–16 (A–D) DNA cleavage reactions in the presence of recognition elements, methyl green (MG), netropsin (Net), and [Co(NH 3 ) 6 ]Cl 3 (Co(III)), where 400 ng pUC19 was initially pre-treated with 8 μM of respective non-covalent binding control at 37° C for 45 min and then with 250 nM, 500 nM, 1 μM, and 2.5 μM test complex in the presence of 1 mM added Na-L-ascorbate at 37°C for 30 min.

    Techniques Used: Incubation, Binding Assay

    17) 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

    18) Product Images from "Comparative Analysis of piggyBac, CRISPR/Cas9 and TALEN Mediated BAC Transgenesis in the Zygote for the Generation of Humanized SIRPA Rats"

    Article Title: Comparative Analysis of piggyBac, CRISPR/Cas9 and TALEN Mediated BAC Transgenesis in the Zygote for the Generation of Humanized SIRPA Rats

    Journal: Scientific Reports

    doi: 10.1038/srep31455

    Strategy for converting hSIRPA -BAC DNA into a piggyBac transposon. ( A ) Diagram illustrating the strategy used for retrofitting hSIRPA-BAC DNA (RP11-887J4) with piggyBac TIR elements. 5′ TIR (green) and 3′ TIR (orange) elements were sub-cloned into pUC19 vector backbone with spectinomycin resistance gene (purple), and 50 bp homology arm sequences (red) used for replacing the chloramphenicol resistance gene in the BAC vector backbone via recombineering technology. The diagram also indicates that the genomic DNA insert in the RP11-887J4 BAC is 176,233 bps, covering the SIRPA genic region, on chromosome 20 between 1,842,086-2,018,318. ( B ) The green arrows indicate the primer pairs used to verify hSIRPA-BAC retrofitting after the recombineering process. ( C ) A schematic diagram describing the transpositioning strategy of hSIRPA-BAC retrofitted with TIR elements mediated by piggyBac transposase. Illustration (i) shows the retrofitted BAC DNA. Illustrations (ii) and (iii) show the process by which the piggyBac transposase proteins bind to the TIR sequences, initiating nicking of the DNA strands, allowing 3′ hydroxyl group at both ends of the transposon to hydrophilic attack the flanking TTAA sequence and freeing the BAC from the spectinomycin resistance gene by forming hairpin structure at the TIR ends. Once the BAC DNA is released from spectinomycin resistance gene, illustration (iv) shows repairing of the linearized BAC DNA by ligating into the complementary TTAA overhangs in the genomic DNA through the mediation of the piggyBac transposase proteins.
    Figure Legend Snippet: Strategy for converting hSIRPA -BAC DNA into a piggyBac transposon. ( A ) Diagram illustrating the strategy used for retrofitting hSIRPA-BAC DNA (RP11-887J4) with piggyBac TIR elements. 5′ TIR (green) and 3′ TIR (orange) elements were sub-cloned into pUC19 vector backbone with spectinomycin resistance gene (purple), and 50 bp homology arm sequences (red) used for replacing the chloramphenicol resistance gene in the BAC vector backbone via recombineering technology. The diagram also indicates that the genomic DNA insert in the RP11-887J4 BAC is 176,233 bps, covering the SIRPA genic region, on chromosome 20 between 1,842,086-2,018,318. ( B ) The green arrows indicate the primer pairs used to verify hSIRPA-BAC retrofitting after the recombineering process. ( C ) A schematic diagram describing the transpositioning strategy of hSIRPA-BAC retrofitted with TIR elements mediated by piggyBac transposase. Illustration (i) shows the retrofitted BAC DNA. Illustrations (ii) and (iii) show the process by which the piggyBac transposase proteins bind to the TIR sequences, initiating nicking of the DNA strands, allowing 3′ hydroxyl group at both ends of the transposon to hydrophilic attack the flanking TTAA sequence and freeing the BAC from the spectinomycin resistance gene by forming hairpin structure at the TIR ends. Once the BAC DNA is released from spectinomycin resistance gene, illustration (iv) shows repairing of the linearized BAC DNA by ligating into the complementary TTAA overhangs in the genomic DNA through the mediation of the piggyBac transposase proteins.

    Techniques Used: BAC Assay, Clone Assay, Plasmid Preparation, Sequencing

    19) Product Images from "The Caulobacter crescentus DNA-(adenine-N6)-methyltransferase CcrM methylates DNA in a distributive manner"

    Article Title: The Caulobacter crescentus DNA-(adenine-N6)-methyltransferase CcrM methylates DNA in a distributive manner

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr768

    Various substrates used for studying CcrM processivity. ( A ) Substrate used by Berdis et al. ( 14 ) to study CcrM processivity, referred to as N 6 60/66-mer. Two GANTC target sites are present, hemimethylated on the upper strand. HindII target sites (GTYRAC) coupled to CcrM target sites were used to screen for methylation on the lower strand. However, only one of the two HindII sites is present, making it impossible to probe the methylation state of the second site. ( B ) The distribution of GANTC sequences (shown as HinfI target sequences) throughout the pUC19 plasmid. The position of each sequence is indicated relative to the plasmid's replication origin. The vector contains a single NdeI target site, which was used in conjunction with HinfI for vector linearization, to facilitate viewing of the progression toward fully methylated state. ( C ) 129-mer substrate containing two CcrM target sites. The expected size of the fragments obtained after HinfI digestion of completely unmethylated, partially methylated and fully methylated substrates are indicated. ( D ) 129-mer_HM substrate used to probe CcrM activity over hemimethylated GANTC sites. A M.TaqI methylation site (TCGA), as well as a HincII restriction site (GTYRAC) were linked to the GANTC site. M.TaqI-established methylation occurs as shown earlier, creating two GANTC sites hemimethylated on the lower strand. CcrM-catalyzed methylation of the upper strand was probed through protection from HincII digestion, which is blocked by hemimethylation.
    Figure Legend Snippet: Various substrates used for studying CcrM processivity. ( A ) Substrate used by Berdis et al. ( 14 ) to study CcrM processivity, referred to as N 6 60/66-mer. Two GANTC target sites are present, hemimethylated on the upper strand. HindII target sites (GTYRAC) coupled to CcrM target sites were used to screen for methylation on the lower strand. However, only one of the two HindII sites is present, making it impossible to probe the methylation state of the second site. ( B ) The distribution of GANTC sequences (shown as HinfI target sequences) throughout the pUC19 plasmid. The position of each sequence is indicated relative to the plasmid's replication origin. The vector contains a single NdeI target site, which was used in conjunction with HinfI for vector linearization, to facilitate viewing of the progression toward fully methylated state. ( C ) 129-mer substrate containing two CcrM target sites. The expected size of the fragments obtained after HinfI digestion of completely unmethylated, partially methylated and fully methylated substrates are indicated. ( D ) 129-mer_HM substrate used to probe CcrM activity over hemimethylated GANTC sites. A M.TaqI methylation site (TCGA), as well as a HincII restriction site (GTYRAC) were linked to the GANTC site. M.TaqI-established methylation occurs as shown earlier, creating two GANTC sites hemimethylated on the lower strand. CcrM-catalyzed methylation of the upper strand was probed through protection from HincII digestion, which is blocked by hemimethylation.

    Techniques Used: Methylation, Plasmid Preparation, Sequencing, Activity Assay

    CcrM processivity assayed using pUC19 ( Figure 1 B) as substrate. A double digestion with HinfI and NdeI was performed to assess the methylation state of the plasmid. pUC19 plasmid linearized by NdeI digestion was used as a control (lane marked C). A large number of incompletely methylated intermediates are formed throughout the duration of the experiment, supporting the conclusion that CcrM is a distributive, rather than a processive methyltransferase. The marker lane (lane marked M) contains the GeneRuler molecular weight marker, provided by Fermentas. The sizes of the major bands are indicated on the left.
    Figure Legend Snippet: CcrM processivity assayed using pUC19 ( Figure 1 B) as substrate. A double digestion with HinfI and NdeI was performed to assess the methylation state of the plasmid. pUC19 plasmid linearized by NdeI digestion was used as a control (lane marked C). A large number of incompletely methylated intermediates are formed throughout the duration of the experiment, supporting the conclusion that CcrM is a distributive, rather than a processive methyltransferase. The marker lane (lane marked M) contains the GeneRuler molecular weight marker, provided by Fermentas. The sizes of the major bands are indicated on the left.

    Techniques Used: Methylation, Plasmid Preparation, Marker, Molecular Weight

    20) 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

    21) Product Images from "Depurination of colibactin-derived interstrand cross-links"

    Article Title: Depurination of colibactin-derived interstrand cross-links

    Journal: bioRxiv

    doi: 10.1101/869313

    DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by denaturing electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, reaction proceed for 1 h, 2 h, 4 h, 6 h, and 9 h. The DNA was isolated, purified, and analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 2 h).
    Figure Legend Snippet: DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by denaturing electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, reaction proceed for 1 h, 2 h, 4 h, 6 h, and 9 h. The DNA was isolated, purified, and analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 2 h).

    Techniques Used: Plasmid Preparation, Cleavage Assay, Electrophoresis, Mutagenesis, Isolation, Purification, Agarose Gel Electrophoresis

    Exposure of pUC19 plasmid DNA to clb + , followed by incubation with Endo IV leads to consumption of undamaged plasmid and formation of nicked and linearized DNA. This is not observed in the clb − or clbL mutant controls. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #4), reacted with buffer (Lane #5), reacted with Endonuclease IV (Lane #6); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #7), reacted with buffer (Lane #8), reacted with Endonuclease IV (Lane #9); circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli (Lane #10), reacted with buffer (Lane #11), reacted with Endonuclease IV (Lane #12). Conditions (Lane #4–#6): circular pUC19 DNA from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane # 4); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #5); the DNA (3.9 µM base pair) was further reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #6). Conditions (Lane #7–#9): circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli. in in M9-CA media for 4 h at 37 °C (Lane #7); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #8); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #9). Conditions (Lane #10–#12): circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli. in M9-CA media for 4 h at 37 °C (Lane #10); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #11); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #12). The DNA was not re-purified and was directly analyzed by native agarose gel electrophoresis (90 V, 1.5 hr).
    Figure Legend Snippet: Exposure of pUC19 plasmid DNA to clb + , followed by incubation with Endo IV leads to consumption of undamaged plasmid and formation of nicked and linearized DNA. This is not observed in the clb − or clbL mutant controls. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #4), reacted with buffer (Lane #5), reacted with Endonuclease IV (Lane #6); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #7), reacted with buffer (Lane #8), reacted with Endonuclease IV (Lane #9); circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli (Lane #10), reacted with buffer (Lane #11), reacted with Endonuclease IV (Lane #12). Conditions (Lane #4–#6): circular pUC19 DNA from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane # 4); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #5); the DNA (3.9 µM base pair) was further reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #6). Conditions (Lane #7–#9): circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli. in in M9-CA media for 4 h at 37 °C (Lane #7); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #8); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #9). Conditions (Lane #10–#12): circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli. in M9-CA media for 4 h at 37 °C (Lane #10); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #11); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #12). The DNA was not re-purified and was directly analyzed by native agarose gel electrophoresis (90 V, 1.5 hr).

    Techniques Used: Plasmid Preparation, Incubation, Mutagenesis, Isolation, Co-Culture Assay, Purification, Agarose Gel Electrophoresis

    Formation of DNA SSBs and DSBs in circular pUC19 plasmid DNA treated with clb + or clb − E. coli . A. SSBs as a function of time. B. DSBs as a function of time. SSBs and DSBs are expressed as intensity of their respective bands relative to an internal control (untreated DNA). Three technical replicates (Fig. S2).
    Figure Legend Snippet: Formation of DNA SSBs and DSBs in circular pUC19 plasmid DNA treated with clb + or clb − E. coli . A. SSBs as a function of time. B. DSBs as a function of time. SSBs and DSBs are expressed as intensity of their respective bands relative to an internal control (untreated DNA). Three technical replicates (Fig. S2).

    Techniques Used: Plasmid Preparation

    Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).
    Figure Legend Snippet: Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).

    Techniques Used: Isolation, Co-Culture Assay, Positive Control, Nucleic Acid Electrophoresis, Cell Culture, Agarose Gel Electrophoresis

    Analysis of induction of AP sites by the colibactin precursor 4. A. Incubation of plasmid pUC19 DNA exposed to 4 in buffer for 18 h results in minor nicking and cleavage. B. Addition of EndoIV increases the amount of nicked and cleaved plasmid. Conditions: A. 5% DMSO was used as vehicle (negative control), and 100 µM cisplatin was used as positive control. DNA ladder (Lane #1); linearized pUC19 DNA standard (Lane #2); 5% DMSO (Lane #3); 100 µM cisplatin (Lane #4); 100 µM 4 (Lane #5); 10 µM 4 (Lane #6); 1 µM 4 (Lane #7); 100 nM 4 (Lane #8); 10 nM 4 (Lane #9). Conditions (Lane #3): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lane #4): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 100 µM cisplatin, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #5–#9): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–10 nM), 5% DMSO, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. The DNA was analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 1.5 h). B. 5% DMSO was used as vehicle. DNA ladder (Lane #1); 5% DMSO (Lane #2); 100 µM 4 (Lane #3); 10 µM 4 (Lane #4); 1 µM 4 (Lane #5); post buffer-reacted after 10 µM 4 (Lane #6); post buffer-reacted after 1 µM 4 (Lane #7); post EndoIV-reacted after 10 µM 4 (Lane #8); post EndoIV-reacted after 1 µM 4 (Lane #9); circular pUC19 plasmid standard (Lane #10); linearized pUC19 plasmid standard (Lane #11). Conditions (Lane #2): circular pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #3–#5): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–1 µM), 5% DMSO, 10 mM citric buffer, pH 5.0, 3 h, 37 °C. Conditions (Lanes #6–#7): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. Conditions (Lanes #8–#9): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), 20 units of Endonuclease IV (New England Biolabs®), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. The DNA was analyzed by native agarose gel electrophoresis (90 V, 2 h).
    Figure Legend Snippet: Analysis of induction of AP sites by the colibactin precursor 4. A. Incubation of plasmid pUC19 DNA exposed to 4 in buffer for 18 h results in minor nicking and cleavage. B. Addition of EndoIV increases the amount of nicked and cleaved plasmid. Conditions: A. 5% DMSO was used as vehicle (negative control), and 100 µM cisplatin was used as positive control. DNA ladder (Lane #1); linearized pUC19 DNA standard (Lane #2); 5% DMSO (Lane #3); 100 µM cisplatin (Lane #4); 100 µM 4 (Lane #5); 10 µM 4 (Lane #6); 1 µM 4 (Lane #7); 100 nM 4 (Lane #8); 10 nM 4 (Lane #9). Conditions (Lane #3): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lane #4): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 100 µM cisplatin, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #5–#9): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–10 nM), 5% DMSO, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. The DNA was analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 1.5 h). B. 5% DMSO was used as vehicle. DNA ladder (Lane #1); 5% DMSO (Lane #2); 100 µM 4 (Lane #3); 10 µM 4 (Lane #4); 1 µM 4 (Lane #5); post buffer-reacted after 10 µM 4 (Lane #6); post buffer-reacted after 1 µM 4 (Lane #7); post EndoIV-reacted after 10 µM 4 (Lane #8); post EndoIV-reacted after 1 µM 4 (Lane #9); circular pUC19 plasmid standard (Lane #10); linearized pUC19 plasmid standard (Lane #11). Conditions (Lane #2): circular pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #3–#5): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–1 µM), 5% DMSO, 10 mM citric buffer, pH 5.0, 3 h, 37 °C. Conditions (Lanes #6–#7): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. Conditions (Lanes #8–#9): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), 20 units of Endonuclease IV (New England Biolabs®), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. The DNA was analyzed by native agarose gel electrophoresis (90 V, 2 h).

    Techniques Used: Incubation, Plasmid Preparation, Negative Control, Positive Control, Agarose Gel Electrophoresis

    DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by neutral gel electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, 1 h, 2 h, 4 h, 6 h, or 9 h. The DNA was isolated, purified, and analyzed by native agarose gel electrophoresis (90 V, 2 h). Order of elution (bottom to top): supercoiled plasmid DNA, linearized DNA, nicked DNA.
    Figure Legend Snippet: DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by neutral gel electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, 1 h, 2 h, 4 h, 6 h, or 9 h. The DNA was isolated, purified, and analyzed by native agarose gel electrophoresis (90 V, 2 h). Order of elution (bottom to top): supercoiled plasmid DNA, linearized DNA, nicked DNA.

    Techniques Used: Plasmid Preparation, Cleavage Assay, Nucleic Acid Electrophoresis, Mutagenesis, Isolation, Purification, Agarose Gel Electrophoresis

    22) Product Images from "Depurination of colibactin-derived interstrand cross-links"

    Article Title: Depurination of colibactin-derived interstrand cross-links

    Journal: bioRxiv

    doi: 10.1101/869313

    DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by denaturing electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, reaction proceed for 1 h, 2 h, 4 h, 6 h, and 9 h. The DNA was isolated, purified, and analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 2 h).
    Figure Legend Snippet: DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by denaturing electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, reaction proceed for 1 h, 2 h, 4 h, 6 h, and 9 h. The DNA was isolated, purified, and analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 2 h).

    Techniques Used: Plasmid Preparation, Cleavage Assay, Electrophoresis, Mutagenesis, Isolation, Purification, Agarose Gel Electrophoresis

    Exposure of pUC19 plasmid DNA to clb + , followed by incubation with Endo IV leads to consumption of undamaged plasmid and formation of nicked and linearized DNA. This is not observed in the clb − or clbL mutant controls. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #4), reacted with buffer (Lane #5), reacted with Endonuclease IV (Lane #6); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #7), reacted with buffer (Lane #8), reacted with Endonuclease IV (Lane #9); circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli (Lane #10), reacted with buffer (Lane #11), reacted with Endonuclease IV (Lane #12). Conditions (Lane #4–#6): circular pUC19 DNA from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane # 4); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #5); the DNA (3.9 µM base pair) was further reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #6). Conditions (Lane #7–#9): circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli. in in M9-CA media for 4 h at 37 °C (Lane #7); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #8); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #9). Conditions (Lane #10–#12): circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli. in M9-CA media for 4 h at 37 °C (Lane #10); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #11); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #12). The DNA was not re-purified and was directly analyzed by native agarose gel electrophoresis (90 V, 1.5 hr).
    Figure Legend Snippet: Exposure of pUC19 plasmid DNA to clb + , followed by incubation with Endo IV leads to consumption of undamaged plasmid and formation of nicked and linearized DNA. This is not observed in the clb − or clbL mutant controls. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #4), reacted with buffer (Lane #5), reacted with Endonuclease IV (Lane #6); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #7), reacted with buffer (Lane #8), reacted with Endonuclease IV (Lane #9); circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli (Lane #10), reacted with buffer (Lane #11), reacted with Endonuclease IV (Lane #12). Conditions (Lane #4–#6): circular pUC19 DNA from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane # 4); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #5); the DNA (3.9 µM base pair) was further reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #6). Conditions (Lane #7–#9): circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli. in in M9-CA media for 4 h at 37 °C (Lane #7); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #8); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #9). Conditions (Lane #10–#12): circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli. in M9-CA media for 4 h at 37 °C (Lane #10); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #11); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #12). The DNA was not re-purified and was directly analyzed by native agarose gel electrophoresis (90 V, 1.5 hr).

    Techniques Used: Plasmid Preparation, Incubation, Mutagenesis, Isolation, Co-Culture Assay, Purification, Agarose Gel Electrophoresis

    Formation of DNA SSBs and DSBs in circular pUC19 plasmid DNA treated with clb + or clb − E. coli . A. SSBs as a function of time. B. DSBs as a function of time. SSBs and DSBs are expressed as intensity of their respective bands relative to an internal control (untreated DNA). Three technical replicates (Fig. S2).
    Figure Legend Snippet: Formation of DNA SSBs and DSBs in circular pUC19 plasmid DNA treated with clb + or clb − E. coli . A. SSBs as a function of time. B. DSBs as a function of time. SSBs and DSBs are expressed as intensity of their respective bands relative to an internal control (untreated DNA). Three technical replicates (Fig. S2).

    Techniques Used: Plasmid Preparation

    Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).
    Figure Legend Snippet: Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).

    Techniques Used: Isolation, Co-Culture Assay, Positive Control, Nucleic Acid Electrophoresis, Cell Culture, Agarose Gel Electrophoresis

    Analysis of induction of AP sites by the colibactin precursor 4. A. Incubation of plasmid pUC19 DNA exposed to 4 in buffer for 18 h results in minor nicking and cleavage. B. Addition of EndoIV increases the amount of nicked and cleaved plasmid. Conditions: A. 5% DMSO was used as vehicle (negative control), and 100 µM cisplatin was used as positive control. DNA ladder (Lane #1); linearized pUC19 DNA standard (Lane #2); 5% DMSO (Lane #3); 100 µM cisplatin (Lane #4); 100 µM 4 (Lane #5); 10 µM 4 (Lane #6); 1 µM 4 (Lane #7); 100 nM 4 (Lane #8); 10 nM 4 (Lane #9). Conditions (Lane #3): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lane #4): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 100 µM cisplatin, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #5–#9): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–10 nM), 5% DMSO, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. The DNA was analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 1.5 h). B. 5% DMSO was used as vehicle. DNA ladder (Lane #1); 5% DMSO (Lane #2); 100 µM 4 (Lane #3); 10 µM 4 (Lane #4); 1 µM 4 (Lane #5); post buffer-reacted after 10 µM 4 (Lane #6); post buffer-reacted after 1 µM 4 (Lane #7); post EndoIV-reacted after 10 µM 4 (Lane #8); post EndoIV-reacted after 1 µM 4 (Lane #9); circular pUC19 plasmid standard (Lane #10); linearized pUC19 plasmid standard (Lane #11). Conditions (Lane #2): circular pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #3–#5): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–1 µM), 5% DMSO, 10 mM citric buffer, pH 5.0, 3 h, 37 °C. Conditions (Lanes #6–#7): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. Conditions (Lanes #8–#9): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), 20 units of Endonuclease IV (New England Biolabs®), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. The DNA was analyzed by native agarose gel electrophoresis (90 V, 2 h).
    Figure Legend Snippet: Analysis of induction of AP sites by the colibactin precursor 4. A. Incubation of plasmid pUC19 DNA exposed to 4 in buffer for 18 h results in minor nicking and cleavage. B. Addition of EndoIV increases the amount of nicked and cleaved plasmid. Conditions: A. 5% DMSO was used as vehicle (negative control), and 100 µM cisplatin was used as positive control. DNA ladder (Lane #1); linearized pUC19 DNA standard (Lane #2); 5% DMSO (Lane #3); 100 µM cisplatin (Lane #4); 100 µM 4 (Lane #5); 10 µM 4 (Lane #6); 1 µM 4 (Lane #7); 100 nM 4 (Lane #8); 10 nM 4 (Lane #9). Conditions (Lane #3): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lane #4): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 100 µM cisplatin, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #5–#9): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–10 nM), 5% DMSO, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. The DNA was analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 1.5 h). B. 5% DMSO was used as vehicle. DNA ladder (Lane #1); 5% DMSO (Lane #2); 100 µM 4 (Lane #3); 10 µM 4 (Lane #4); 1 µM 4 (Lane #5); post buffer-reacted after 10 µM 4 (Lane #6); post buffer-reacted after 1 µM 4 (Lane #7); post EndoIV-reacted after 10 µM 4 (Lane #8); post EndoIV-reacted after 1 µM 4 (Lane #9); circular pUC19 plasmid standard (Lane #10); linearized pUC19 plasmid standard (Lane #11). Conditions (Lane #2): circular pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #3–#5): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–1 µM), 5% DMSO, 10 mM citric buffer, pH 5.0, 3 h, 37 °C. Conditions (Lanes #6–#7): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. Conditions (Lanes #8–#9): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), 20 units of Endonuclease IV (New England Biolabs®), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. The DNA was analyzed by native agarose gel electrophoresis (90 V, 2 h).

    Techniques Used: Incubation, Plasmid Preparation, Negative Control, Positive Control, Agarose Gel Electrophoresis

    DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by neutral gel electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, 1 h, 2 h, 4 h, 6 h, or 9 h. The DNA was isolated, purified, and analyzed by native agarose gel electrophoresis (90 V, 2 h). Order of elution (bottom to top): supercoiled plasmid DNA, linearized DNA, nicked DNA.
    Figure Legend Snippet: DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by neutral gel electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, 1 h, 2 h, 4 h, 6 h, or 9 h. The DNA was isolated, purified, and analyzed by native agarose gel electrophoresis (90 V, 2 h). Order of elution (bottom to top): supercoiled plasmid DNA, linearized DNA, nicked DNA.

    Techniques Used: Plasmid Preparation, Cleavage Assay, Nucleic Acid Electrophoresis, Mutagenesis, Isolation, Purification, Agarose Gel Electrophoresis

    23) Product Images from "Di-copper metallodrugs promote NCI-60 chemotherapy via singlet oxygen and superoxide production with tandem TA/TA and AT/AT oligonucleotide discrimination"

    Article Title: Di-copper metallodrugs promote NCI-60 chemotherapy via singlet oxygen and superoxide production with tandem TA/TA and AT/AT oligonucleotide discrimination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky105

    ( A ) Mean tail moments of untreated SKOV-3 cells and 1.0 μM Cu-Oda, Cu-Terph and Dox. ( B ) Topoisomerase I unwinding across concentration range 0.10–400 μM for Cu-Oda and Cu-Terph . ( C ). DSBs induced by Cu-Oda and Cu-Terph , detected by immunostaining of γH2AX with MFI presented for di -Cu 2+ complexes and Dox. ( D ) CD spectra of Cu-Oda with stDNA and alternating co-polymers poly[d(A⋅T) 2 ] and poly[d(G⋅C) 2 ] (100 μM) at loading ratios 0.01–0.075.
    Figure Legend Snippet: ( A ) Mean tail moments of untreated SKOV-3 cells and 1.0 μM Cu-Oda, Cu-Terph and Dox. ( B ) Topoisomerase I unwinding across concentration range 0.10–400 μM for Cu-Oda and Cu-Terph . ( C ). DSBs induced by Cu-Oda and Cu-Terph , detected by immunostaining of γH2AX with MFI presented for di -Cu 2+ complexes and Dox. ( D ) CD spectra of Cu-Oda with stDNA and alternating co-polymers poly[d(A⋅T) 2 ] and poly[d(G⋅C) 2 ] (100 μM) at loading ratios 0.01–0.075.

    Techniques Used: Concentration Assay, Immunostaining

    24) Product Images from "Bacterial rhomboid proteases mediate quality control of orphan membrane proteins"

    Article Title: Bacterial rhomboid proteases mediate quality control of orphan membrane proteins

    Journal: The EMBO Journal

    doi: 10.15252/embj.2019102922

    GlpG specifically targets orphan HybA and does not influence hydrogenase‐2 activity Bacterial two‐hybrid analysis with HybA and/or HybB fused chromosomally with the T25 and T18 domains of B. pertussis CyaA, respectively, in the absence of endogenous CyaA. Bacteria were grown on LB agar containing 20 μg/ml X‐gal at 37°C for 14 h in the presence/absence of O 2 . Scale bar, 1 cm. Western blot analysis to detect HybA cleavage in S. sonnei Δ rhom7 by wild‐type (+) or inactive (S 201 A) GlpG expressed chromosomally (native) or from pUC19 (plasmid) with (+)/without (−) HybB. HybA was expressed from its native locus or a plasmid (pHybA). Quantification of the ratio of cleaved/uncleaved HybA in strains with (+) or without (−) HybB. Mean ± S.D. of three experiments. * P
    Figure Legend Snippet: GlpG specifically targets orphan HybA and does not influence hydrogenase‐2 activity Bacterial two‐hybrid analysis with HybA and/or HybB fused chromosomally with the T25 and T18 domains of B. pertussis CyaA, respectively, in the absence of endogenous CyaA. Bacteria were grown on LB agar containing 20 μg/ml X‐gal at 37°C for 14 h in the presence/absence of O 2 . Scale bar, 1 cm. Western blot analysis to detect HybA cleavage in S. sonnei Δ rhom7 by wild‐type (+) or inactive (S 201 A) GlpG expressed chromosomally (native) or from pUC19 (plasmid) with (+)/without (−) HybB. HybA was expressed from its native locus or a plasmid (pHybA). Quantification of the ratio of cleaved/uncleaved HybA in strains with (+) or without (−) HybB. Mean ± S.D. of three experiments. * P

    Techniques Used: Activity Assay, Western Blot, Plasmid Preparation

    Rhomboids prevent aggregation of orphan substrates in the inner membrane Western blot analysis probing the localisation and status of plasmid‐encoded N‐terminally V5‐tagged wild‐type (WT) or modified (P 300 A) HybA in S. sonnei Δ rhom7 Δ hybB with wild‐type (+) or inactive (SAHA) GlpG expressed from pUC19. Whole‐cell lysate (Whole cell.), Soluble (Sol.), Membrane (detergent‐solubilised, Mem.) and the Aggregate (Agg.) fractions are shown. HybA that is uncleaved, cleaved only by GlpG and further degraded post‐GlpG cleavage is marked by black, red and green arrows, respectively. Model of rhomboid‐mediated quality control by selectively targeting orphan components of multiprotein respiratory complexes. Source data are available online for this figure.
    Figure Legend Snippet: Rhomboids prevent aggregation of orphan substrates in the inner membrane Western blot analysis probing the localisation and status of plasmid‐encoded N‐terminally V5‐tagged wild‐type (WT) or modified (P 300 A) HybA in S. sonnei Δ rhom7 Δ hybB with wild‐type (+) or inactive (SAHA) GlpG expressed from pUC19. Whole‐cell lysate (Whole cell.), Soluble (Sol.), Membrane (detergent‐solubilised, Mem.) and the Aggregate (Agg.) fractions are shown. HybA that is uncleaved, cleaved only by GlpG and further degraded post‐GlpG cleavage is marked by black, red and green arrows, respectively. Model of rhomboid‐mediated quality control by selectively targeting orphan components of multiprotein respiratory complexes. Source data are available online for this figure.

    Techniques Used: Western Blot, Plasmid Preparation, Modification

    Rhomboid cleavage licenses further degradation of substrates Western blot analysis (probing with an anti‐V5 mAb) to detect degradation of N‐terminally V5‐tagged wild‐type (WT) or modified (P 300 A) HybA in S. sonnei Δ rhom7 chromosomally expressing wild‐type (+) or inactive (S 201 A) GlpG with (+) or without (−) HybB. Degradation of V5‐tagged HybA at times after blocking protein translation at T 0 in the presence (+) or absence (−) of HybB. Western blot analysis of N‐terminally V5‐tagged wild‐type (WT) or modified (P 259 A) FdoH in S. sonnei Δ rhom7 chromosomally expressing wild‐type (+) or inactive (S 201 A) GlpG with (+)/without (−) FdoI. Western blot analysis of N‐terminally V5‐tagged wild‐type (WT) or modified (P 259 A) FdnH in S. sonnei Δ rhom7 with wild‐type (+) or inactive (SAHA) Rhom7 expressed from pBAD33 with (+)/without (−) FdnI. Degradation of N‐terminally V5‐tagged wild‐type (WT) or modified (P 259 A) FdoH (E) or FdnH (F) in S. sonnei Δ rhom7 with wild‐type (+) or inactive (S 201 A, SAHA) GlpG expressed chromosomally (native) or from pUC19 (plasmid) without FdoI or FdnI (−), respectively, +/− exposure to 400 μM CuCl 2 for 30 min. Data information: Rhomboid substrates that are uncleaved, cleaved by GlpG or cleaved by Rhom7 are marked by black, red and blue arrows, respectively. Degradation products post‐rhomboid cleavage are marked by green arrows. RecA, loading control. Source data are available online for this figure.
    Figure Legend Snippet: Rhomboid cleavage licenses further degradation of substrates Western blot analysis (probing with an anti‐V5 mAb) to detect degradation of N‐terminally V5‐tagged wild‐type (WT) or modified (P 300 A) HybA in S. sonnei Δ rhom7 chromosomally expressing wild‐type (+) or inactive (S 201 A) GlpG with (+) or without (−) HybB. Degradation of V5‐tagged HybA at times after blocking protein translation at T 0 in the presence (+) or absence (−) of HybB. Western blot analysis of N‐terminally V5‐tagged wild‐type (WT) or modified (P 259 A) FdoH in S. sonnei Δ rhom7 chromosomally expressing wild‐type (+) or inactive (S 201 A) GlpG with (+)/without (−) FdoI. Western blot analysis of N‐terminally V5‐tagged wild‐type (WT) or modified (P 259 A) FdnH in S. sonnei Δ rhom7 with wild‐type (+) or inactive (SAHA) Rhom7 expressed from pBAD33 with (+)/without (−) FdnI. Degradation of N‐terminally V5‐tagged wild‐type (WT) or modified (P 259 A) FdoH (E) or FdnH (F) in S. sonnei Δ rhom7 with wild‐type (+) or inactive (S 201 A, SAHA) GlpG expressed chromosomally (native) or from pUC19 (plasmid) without FdoI or FdnI (−), respectively, +/− exposure to 400 μM CuCl 2 for 30 min. Data information: Rhomboid substrates that are uncleaved, cleaved by GlpG or cleaved by Rhom7 are marked by black, red and blue arrows, respectively. Degradation products post‐rhomboid cleavage are marked by green arrows. RecA, loading control. Source data are available online for this figure.

    Techniques Used: Western Blot, Modification, Expressing, Blocking Assay, Plasmid Preparation

    25) 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

    26) 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

    27) 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

    28) Product Images from "Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv738

    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.
    Figure Legend 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.

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

    29) Product Images from "Depurination of colibactin-derived interstrand cross-links"

    Article Title: Depurination of colibactin-derived interstrand cross-links

    Journal: bioRxiv

    doi: 10.1101/869313

    DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by denaturing electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, reaction proceed for 1 h, 2 h, 4 h, 6 h, and 9 h. The DNA was isolated, purified, and analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 2 h).
    Figure Legend Snippet: DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by denaturing electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, reaction proceed for 1 h, 2 h, 4 h, 6 h, and 9 h. The DNA was isolated, purified, and analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 2 h).

    Techniques Used: Plasmid Preparation, Cleavage Assay, Electrophoresis, Mutagenesis, Isolation, Purification, Agarose Gel Electrophoresis

    Exposure of pUC19 plasmid DNA to clb + , followed by incubation with Endo IV leads to consumption of undamaged plasmid and formation of nicked and linearized DNA. This is not observed in the clb − or clbL mutant controls. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #4), reacted with buffer (Lane #5), reacted with Endonuclease IV (Lane #6); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #7), reacted with buffer (Lane #8), reacted with Endonuclease IV (Lane #9); circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli (Lane #10), reacted with buffer (Lane #11), reacted with Endonuclease IV (Lane #12). Conditions (Lane #4–#6): circular pUC19 DNA from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane # 4); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #5); the DNA (3.9 µM base pair) was further reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #6). Conditions (Lane #7–#9): circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli. in in M9-CA media for 4 h at 37 °C (Lane #7); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #8); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #9). Conditions (Lane #10–#12): circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli. in M9-CA media for 4 h at 37 °C (Lane #10); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #11); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #12). The DNA was not re-purified and was directly analyzed by native agarose gel electrophoresis (90 V, 1.5 hr).
    Figure Legend Snippet: Exposure of pUC19 plasmid DNA to clb + , followed by incubation with Endo IV leads to consumption of undamaged plasmid and formation of nicked and linearized DNA. This is not observed in the clb − or clbL mutant controls. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #4), reacted with buffer (Lane #5), reacted with Endonuclease IV (Lane #6); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #7), reacted with buffer (Lane #8), reacted with Endonuclease IV (Lane #9); circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli (Lane #10), reacted with buffer (Lane #11), reacted with Endonuclease IV (Lane #12). Conditions (Lane #4–#6): circular pUC19 DNA from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane # 4); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #5); the DNA (3.9 µM base pair) was further reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #6). Conditions (Lane #7–#9): circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli. in in M9-CA media for 4 h at 37 °C (Lane #7); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1 (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #8); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #9). Conditions (Lane #10–#12): circular pUC19 DNA isolated from co-culture with clbL mutant (S179A) BW25113 E. coli. in M9-CA media for 4 h at 37 °C (Lane #10); the DNA (3.9 µM base pair) was reacted with NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #11); the DNA (3.9 µM base pair) was reacted with 20 units of Endonuclease IV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 20 hours (Lane #12). The DNA was not re-purified and was directly analyzed by native agarose gel electrophoresis (90 V, 1.5 hr).

    Techniques Used: Plasmid Preparation, Incubation, Mutagenesis, Isolation, Co-Culture Assay, Purification, Agarose Gel Electrophoresis

    Formation of DNA SSBs and DSBs in circular pUC19 plasmid DNA treated with clb + or clb − E. coli . A. SSBs as a function of time. B. DSBs as a function of time. SSBs and DSBs are expressed as intensity of their respective bands relative to an internal control (untreated DNA). Three technical replicates (Fig. S2).
    Figure Legend Snippet: Formation of DNA SSBs and DSBs in circular pUC19 plasmid DNA treated with clb + or clb − E. coli . A. SSBs as a function of time. B. DSBs as a function of time. SSBs and DSBs are expressed as intensity of their respective bands relative to an internal control (untreated DNA). Three technical replicates (Fig. S2).

    Techniques Used: Plasmid Preparation

    Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).
    Figure Legend Snippet: Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).

    Techniques Used: Isolation, Co-Culture Assay, Positive Control, Nucleic Acid Electrophoresis, Cell Culture, Agarose Gel Electrophoresis

    Analysis of induction of AP sites by the colibactin precursor 4. A. Incubation of plasmid pUC19 DNA exposed to 4 in buffer for 18 h results in minor nicking and cleavage. B. Addition of EndoIV increases the amount of nicked and cleaved plasmid. Conditions: A. 5% DMSO was used as vehicle (negative control), and 100 µM cisplatin was used as positive control. DNA ladder (Lane #1); linearized pUC19 DNA standard (Lane #2); 5% DMSO (Lane #3); 100 µM cisplatin (Lane #4); 100 µM 4 (Lane #5); 10 µM 4 (Lane #6); 1 µM 4 (Lane #7); 100 nM 4 (Lane #8); 10 nM 4 (Lane #9). Conditions (Lane #3): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lane #4): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 100 µM cisplatin, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #5–#9): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–10 nM), 5% DMSO, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. The DNA was analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 1.5 h). B. 5% DMSO was used as vehicle. DNA ladder (Lane #1); 5% DMSO (Lane #2); 100 µM 4 (Lane #3); 10 µM 4 (Lane #4); 1 µM 4 (Lane #5); post buffer-reacted after 10 µM 4 (Lane #6); post buffer-reacted after 1 µM 4 (Lane #7); post EndoIV-reacted after 10 µM 4 (Lane #8); post EndoIV-reacted after 1 µM 4 (Lane #9); circular pUC19 plasmid standard (Lane #10); linearized pUC19 plasmid standard (Lane #11). Conditions (Lane #2): circular pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #3–#5): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–1 µM), 5% DMSO, 10 mM citric buffer, pH 5.0, 3 h, 37 °C. Conditions (Lanes #6–#7): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. Conditions (Lanes #8–#9): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), 20 units of Endonuclease IV (New England Biolabs®), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. The DNA was analyzed by native agarose gel electrophoresis (90 V, 2 h).
    Figure Legend Snippet: Analysis of induction of AP sites by the colibactin precursor 4. A. Incubation of plasmid pUC19 DNA exposed to 4 in buffer for 18 h results in minor nicking and cleavage. B. Addition of EndoIV increases the amount of nicked and cleaved plasmid. Conditions: A. 5% DMSO was used as vehicle (negative control), and 100 µM cisplatin was used as positive control. DNA ladder (Lane #1); linearized pUC19 DNA standard (Lane #2); 5% DMSO (Lane #3); 100 µM cisplatin (Lane #4); 100 µM 4 (Lane #5); 10 µM 4 (Lane #6); 1 µM 4 (Lane #7); 100 nM 4 (Lane #8); 10 nM 4 (Lane #9). Conditions (Lane #3): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lane #4): linearized pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 100 µM cisplatin, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #5–#9): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–10 nM), 5% DMSO, 10 mM citric buffer, pH 5.0, 4 h, 37 °C. The DNA was analyzed by 0.4% NaOH denaturing agarose gel electrophoresis (90 V, 1.5 h). B. 5% DMSO was used as vehicle. DNA ladder (Lane #1); 5% DMSO (Lane #2); 100 µM 4 (Lane #3); 10 µM 4 (Lane #4); 1 µM 4 (Lane #5); post buffer-reacted after 10 µM 4 (Lane #6); post buffer-reacted after 1 µM 4 (Lane #7); post EndoIV-reacted after 10 µM 4 (Lane #8); post EndoIV-reacted after 1 µM 4 (Lane #9); circular pUC19 plasmid standard (Lane #10); linearized pUC19 plasmid standard (Lane #11). Conditions (Lane #2): circular pUC19 DNA (15.4 µM in base pairs), 5% DMSO (vehicle), 10 mM citric buffer, pH 5.0, 4 h, 37 °C. Conditions (Lanes #3–#5): circular pUC19 DNA (15.4 µM in base pairs), 4 (100 µM–1 µM), 5% DMSO, 10 mM citric buffer, pH 5.0, 3 h, 37 °C. Conditions (Lanes #6–#7): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. Conditions (Lanes #8–#9): 4 (10 µM–1 µM)-treated circular pUC19 DNA (3.9 µM in base pairs), 20 units of Endonuclease IV (New England Biolabs®), NEBuffer 3.1® (New England Biolabs®), pH 7.9, at 37 °C for 18 h. The DNA was analyzed by native agarose gel electrophoresis (90 V, 2 h).

    Techniques Used: Incubation, Plasmid Preparation, Negative Control, Positive Control, Agarose Gel Electrophoresis

    DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by neutral gel electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, 1 h, 2 h, 4 h, 6 h, or 9 h. The DNA was isolated, purified, and analyzed by native agarose gel electrophoresis (90 V, 2 h). Order of elution (bottom to top): supercoiled plasmid DNA, linearized DNA, nicked DNA.
    Figure Legend Snippet: DNA plasmid cleavage assay employing circular pUC19 DNA and E. coli and analysis by neutral gel electrophoresis. DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); clb − BW25113 E. coli 1 h (Lane #4), 2 h (Lane #5), 4 h (Lane #6), 6 h (Lane #7), and 9 h (Lane #8); clb + BW25113 E. coli 1 h (Lane #9), 2 h (Lane #10), 4 h (Lane #11), 6 h (Lane #12), and 9 h (Lane #13); clbL mutant (S179A) BW25113 E. coli 1 h (Lane #14), 2 h (Lane #15), 4 h (Lane #16), 6 h (Lane #17), and 9 h (Lane #18). Conditions (Lane #4–#18): clb − BW25113 E. coli (Lane #4–#8), clb + BW25113 E. coli (Lane #9–#13), and clbL mutant (S179A) BW25113 E. coli (Lane #14–#18), circular pUC19 DNA (7.7 µM in base pairs), M9-CA media, 37 °C, 1 h, 2 h, 4 h, 6 h, or 9 h. The DNA was isolated, purified, and analyzed by native agarose gel electrophoresis (90 V, 2 h). Order of elution (bottom to top): supercoiled plasmid DNA, linearized DNA, nicked DNA.

    Techniques Used: Plasmid Preparation, Cleavage Assay, Nucleic Acid Electrophoresis, Mutagenesis, Isolation, Purification, Agarose Gel Electrophoresis

    30) 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

    31) Product Images from "Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv738

    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.
    Figure Legend 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.

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

    Related Articles

    Clone Assay:

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: .. 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: .. 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: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
    Article Snippet: .. To generate the 4mC spike-in control, 0.5 ng of pUC19 vector (NEB) was PCR amplified as follows in a 50 μl reaction: 2.5 U RedTaq polymerase (Sigma), 5 μl 10× reaction buffer, 1 μl 4mdCTP (Trilink)/dATP/dGTP/dTTP cocktail (10 mM each), 1 μl 10 mM forward primer (5′-GCGGTAATACGGTTATCCAC), 1 μl 10 mM reverse primer (5′-TGCTGATAAATCTGGAGCCG). ..

    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.

    Positive Control:

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: .. Two control experiments were also conducted: a positive control consisting of electrocompetent ΔcysE cells transformed with cysE in pUC19 vector and the other with electrocompetent ΔcysKΔcysM cells transformed with codon optimized cysM in pUC19 vector. .. The second positive control group supplemented the lack of cysteine through growing the knockout cells with empty pUC19 vectors on a fully supplemented media, LB + Amp, and were allowed to incubate at 37 °C overnight.

    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.

    Polymerase Chain Reaction:

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

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

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
    Article Snippet: .. To generate the 4mC spike-in control, 0.5 ng of pUC19 vector (NEB) was PCR amplified as follows in a 50 μl reaction: 2.5 U RedTaq polymerase (Sigma), 5 μl 10× reaction buffer, 1 μl 4mdCTP (Trilink)/dATP/dGTP/dTTP cocktail (10 mM each), 1 μl 10 mM forward primer (5′-GCGGTAATACGGTTATCCAC), 1 μl 10 mM reverse primer (5′-TGCTGATAAATCTGGAGCCG). ..

    Transformation Assay:

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

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: .. Two control experiments were also conducted: a positive control consisting of electrocompetent ΔcysE cells transformed with cysE in pUC19 vector and the other with electrocompetent ΔcysKΔcysM cells transformed with codon optimized cysM in pUC19 vector. .. The second positive control group supplemented the lack of cysteine through growing the knockout cells with empty pUC19 vectors on a fully supplemented media, LB + Amp, and were allowed to incubate at 37 °C overnight.

    Plasmid Preparation:

    Article Title: Synthesis of the unnatural amino acid Nα-Nε-(ferrocene-1-acetyl)-l-lysine: a novel organometallic nuclease
    Article Snippet: .. DNA cleavage assays (20 μl) contained 1.0 μg of pUC19 vector DNA (New England Biolabs, 75 μM bp) along with increasing concentrations (0 - 150 μM) of 1 or ferroceneacetic acid (Sigma) dissolved in 10% DMSO in 10 mM Tris-HCl, pH 8.0. .. After incubation for 16 h at 25 °C, the cleavage products of each reaction were separated by gel electrophoresis (1% agarose, 0.5 μg/mL ethidium bromide) in 1×TBE buffer at 90 V for 90 min, visualized by UV light, and captured on a digital image.

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

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

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
    Article Snippet: .. To generate the 4mC spike-in control, 0.5 ng of pUC19 vector (NEB) was PCR amplified as follows in a 50 μl reaction: 2.5 U RedTaq polymerase (Sigma), 5 μl 10× reaction buffer, 1 μl 4mdCTP (Trilink)/dATP/dGTP/dTTP cocktail (10 mM each), 1 μl 10 mM forward primer (5′-GCGGTAATACGGTTATCCAC), 1 μl 10 mM reverse primer (5′-TGCTGATAAATCTGGAGCCG). ..

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: .. Two control experiments were also conducted: a positive control consisting of electrocompetent ΔcysE cells transformed with cysE in pUC19 vector and the other with electrocompetent ΔcysKΔcysM cells transformed with codon optimized cysM in pUC19 vector. .. The second positive control group supplemented the lack of cysteine through growing the knockout cells with empty pUC19 vectors on a fully supplemented media, LB + Amp, and were allowed to incubate at 37 °C overnight.

    Article Title: Depurination of colibactin-derived interstrand cross-links
    Article Snippet: .. The pUC19 DNA exposed to synthetic compounds in plasmid cleavage or DNA cross-linking assays was directly diluted and used for EndoIV stability tests. .. To set up each reaction, 50 ng of processed DNA was mixed with 20 units of EndoIV in NEBuffer 3.1® (New England Biolabs®), pH 7.9, in a total volume of 20 µL for 16 h−20 h (unless otherwise noted) at 37 °C.

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    New England Biolabs puc19 vector
    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 <t>pUC19</t> 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.
    Puc19 Vector, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-018-19920-y

    Figure Lengend 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.

    Article Snippet: 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).

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

    Lane 1–4 (A–D) DNA cleavage reactions with 250 nM, 500 nM, 1.0 μM, and 2.5 μM test complex (A) Cu-Phen, (B) Cu-DPQ-Phen, (C) Cu-DPPZ-Phen, and (D) Cu-Terph, 400 ng superhelical pUC19 and 1 mM added Na-L-ascorbate incubated at 37°C for 30 min . Lanes 5–16 (A–D) DNA cleavage reactions in the presence of recognition elements, methyl green (MG), netropsin (Net), and [Co(NH 3 ) 6 ]Cl 3 (Co(III)), where 400 ng pUC19 was initially pre-treated with 8 μM of respective non-covalent binding control at 37° C for 45 min and then with 250 nM, 500 nM, 1 μM, and 2.5 μM test complex in the presence of 1 mM added Na-L-ascorbate at 37°C for 30 min.

    Journal: Frontiers in Chemistry

    Article Title: DNA oxidation profiles of copper phenanthrene chemical nucleases

    doi: 10.3389/fchem.2015.00028

    Figure Lengend Snippet: Lane 1–4 (A–D) DNA cleavage reactions with 250 nM, 500 nM, 1.0 μM, and 2.5 μM test complex (A) Cu-Phen, (B) Cu-DPQ-Phen, (C) Cu-DPPZ-Phen, and (D) Cu-Terph, 400 ng superhelical pUC19 and 1 mM added Na-L-ascorbate incubated at 37°C for 30 min . Lanes 5–16 (A–D) DNA cleavage reactions in the presence of recognition elements, methyl green (MG), netropsin (Net), and [Co(NH 3 ) 6 ]Cl 3 (Co(III)), where 400 ng pUC19 was initially pre-treated with 8 μM of respective non-covalent binding control at 37° C for 45 min and then with 250 nM, 500 nM, 1 μM, and 2.5 μM test complex in the presence of 1 mM added Na-L-ascorbate at 37°C for 30 min.

    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, 1 mM Na-L-ascorbate, 400 ng superhelical pUC19 (NEB, N3041) and varying concentrations of test complex (250 nM, 500 nM, 1 μM and 2.5 μM).

    Techniques: Incubation, Binding Assay

    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