asei  (New England Biolabs)


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    New England Biolabs asei
    Asei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/asei/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    asei - by Bioz Stars, 2022-05
    97/100 stars

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    New England Biolabs nebuffer 3 1
    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). Linear-denat. = DSB/linearized DNA in denaturing form, linear-XL = DSB/linerized DNA cross-linked by colibactin or cisplatin, SC = supercoiled, nicked = SSB, linear = DSB.
    Nebuffer 3 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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). Linear-denat. = DSB/linearized DNA in denaturing form, linear-XL = DSB/linerized DNA cross-linked by colibactin or cisplatin, SC = supercoiled, nicked = SSB, linear = DSB.

    Journal: Biochemistry

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

    doi: 10.1021/acs.biochem.9b01070

    Figure Lengend 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). Linear-denat. = DSB/linearized DNA in denaturing form, linear-XL = DSB/linerized DNA cross-linked by colibactin or cisplatin, SC = supercoiled, nicked = SSB, linear = DSB.

    Article Snippet: 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.

    Techniques: Incubation, Plasmid Preparation, Negative Control, Positive Control, 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).

    Journal: Biochemistry

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

    doi: 10.1021/acs.biochem.9b01070

    Figure Lengend 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).

    Article Snippet: 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.

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

    smFISH and smiFISH in C. elegans embryos A . Schematic illustration of smFISH probes. B . Schematic illustration of smiFISH probes. C . nos-2 RNA was visualized using smiFISH (magenta) and smFISH (green). nos-2 smiFISH primary probes used FLAP-Y sequences and the secondary FLAP-Y probe was 5’ and 3’ dual-conjugated with Quasar 670 fluorophores. nos-2 smFISH probes were 3’ single-conjugated with Cal Fluor 610. D . imb-2 RNA was visualized using smFISH (magenta) and smiFISH (green). imb-2 smFISH probes were 3’ single-conjugated with Quasar 670 fluorophores. imb-2 smiFISH primary probes used FLAP-Y sequences and the secondary FLAP-Y probe was 5’ and 3’ dual-conjugated with Cal Fluor 610. Embryos were counterstained with DAPI in blue (C, D) . Three biological replicates were performed for each experiment using newly annealed smiFISH probes for each replicate. Scale bars represent 10 um.

    Journal: bioRxiv

    Article Title: Improved methods for protein and single-molecule RNA detection in C. elegans embryos

    doi: 10.1101/2021.05.07.443170

    Figure Lengend Snippet: smFISH and smiFISH in C. elegans embryos A . Schematic illustration of smFISH probes. B . Schematic illustration of smiFISH probes. C . nos-2 RNA was visualized using smiFISH (magenta) and smFISH (green). nos-2 smiFISH primary probes used FLAP-Y sequences and the secondary FLAP-Y probe was 5’ and 3’ dual-conjugated with Quasar 670 fluorophores. nos-2 smFISH probes were 3’ single-conjugated with Cal Fluor 610. D . imb-2 RNA was visualized using smFISH (magenta) and smiFISH (green). imb-2 smFISH probes were 3’ single-conjugated with Quasar 670 fluorophores. imb-2 smiFISH primary probes used FLAP-Y sequences and the secondary FLAP-Y probe was 5’ and 3’ dual-conjugated with Cal Fluor 610. Embryos were counterstained with DAPI in blue (C, D) . Three biological replicates were performed for each experiment using newly annealed smiFISH probes for each replicate. Scale bars represent 10 um.

    Article Snippet: New England Bio Labs Buffer 3 (or 3.1) (NEB cat. no. B7203S). smiFISH probe annealing: Combine primary probes at equimolar ratio and dilute to 0.833 uM in Tris pH 8.0.

    Techniques:

    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).

    Journal: bioRxiv

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

    doi: 10.1101/869313

    Figure Lengend 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).

    Article Snippet: The NEBuffer 3.1® (New England Biolabs®) contained 100 mM sodium chloride, 50 mM Tris-HCl, 10 mM magnesium chloride, and 100 µg/ml BSA.

    Techniques: Plasmid Preparation, Incubation, Mutagenesis, Isolation, Co-Culture Assay, Purification, 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).

    Journal: bioRxiv

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

    doi: 10.1101/869313

    Figure Lengend 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).

    Article Snippet: The NEBuffer 3.1® (New England Biolabs®) contained 100 mM sodium chloride, 50 mM Tris-HCl, 10 mM magnesium chloride, and 100 µg/ml BSA.

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