nebuffer 3 1  (New England Biolabs)


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    Name:
    NEBuffer 3 1
    Description:

    Catalog Number:
    B7203
    Price:
    25
    Category:
    Buffers Loading Dyes Diluents
    Applications:
    DNA Manipulation
    Size:
    5 ml
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    New England Biolabs nebuffer 3 1
    NEBuffer 3 1

    https://www.bioz.com/result/nebuffer 3 1/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nebuffer 3 1 - by Bioz Stars, 2021-07
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    1) Product Images from "Depurination of colibactin-derived interstrand cross-links."

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

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.9b01070

    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.
    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). 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.

    Techniques Used: 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).
    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

    2) 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

    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

    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

    3) 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

    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

    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

    4) 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

    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

    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

    5) Product Images from "Improved methods for protein and single-molecule RNA detection in C. elegans embryos"

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

    Journal: bioRxiv

    doi: 10.1101/2021.05.07.443170

    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.
    Figure Legend 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.

    Techniques Used:

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

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

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.9b01070

    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.
    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). 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.

    Techniques Used: 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).
    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

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

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

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.9b01070

    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.
    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). 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.

    Techniques Used: 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).
    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

    8) Product Images from "CRISPR/dCas9-mediated biosensor for detection of tick-borne diseases"

    Article Title: CRISPR/dCas9-mediated biosensor for detection of tick-borne diseases

    Journal: Sensors and Actuators. B, Chemical

    doi: 10.1016/j.snb.2018.06.069

    Ability of dCas9 ribonucleoprotein (RNP) in reaction buffer. (A)  in vitro  cleavage assay to investigate the activity of gRNAs in the RPA buffer condition. Cas9 RNP could cleave the PCR products in both the RPA buffer and the NEBuffer 3.1 condition only when gRNAs were matched to the target PCR products. (B) Electrophoretic mobility shift assay (EMSA) using dCas9 RNP and the 5′ biotinylated DNA duplexes. The target DNA duplexes were only shifted with the matched gRNAs in both the RPA buffer and the NEBuffer 3.1 condition.
    Figure Legend Snippet: Ability of dCas9 ribonucleoprotein (RNP) in reaction buffer. (A) in vitro cleavage assay to investigate the activity of gRNAs in the RPA buffer condition. Cas9 RNP could cleave the PCR products in both the RPA buffer and the NEBuffer 3.1 condition only when gRNAs were matched to the target PCR products. (B) Electrophoretic mobility shift assay (EMSA) using dCas9 RNP and the 5′ biotinylated DNA duplexes. The target DNA duplexes were only shifted with the matched gRNAs in both the RPA buffer and the NEBuffer 3.1 condition.

    Techniques Used: In Vitro, Cleavage Assay, Activity Assay, Recombinase Polymerase Amplification, Polymerase Chain Reaction, Electrophoretic Mobility Shift Assay

    Related Articles

    Negative Control:

    Article Title: Depurination of colibactin-derived interstrand cross-links.
    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. .. To set up each negative control, 50 ng of processed DNA was mixed with NEBuffer 3.1® (New England Biolabs®), pH 7.9, in a total volume of 20 μL for 16 h−20 h (otherwise otherwise noted) at 37 °C. ..

    Article Title: Depurination of colibactin-derived interstrand cross-links
    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. .. To set up each negative control, 50 ng of processed DNA was mixed with NEBuffer 3.1® (New England Biolabs®), pH 7.9, in a total volume of 20 µL for 16 h−20 h (otherwise otherwise noted) at 37 °C. ..

    Positive Control:

    Article Title: CRISPR/dCas9-mediated biosensor for detection of tick-borne diseases
    Article Snippet: In 10 μl reactions, buffer mixed PCR products were incubated with 1 μg Cas9 protein and 750 ng sgRNA for 1 h at 37 °C as previous study [ ]. .. For the positive control of the cleavage assay, same PCR products were cleaved in 1× NEBuffer 3.1 (100 mM NaCl, 50 mM Tris−HCl, 10 mM MgCl2 , 100 μg/ml BSA, New England BioLabs) condition. .. The RNase A (4 μg) was added to samples to remove sgRNA and the final samples were analyzed with agarose gel electrophoresis.

    Cleavage Assay:

    Article Title: CRISPR/dCas9-mediated biosensor for detection of tick-borne diseases
    Article Snippet: In 10 μl reactions, buffer mixed PCR products were incubated with 1 μg Cas9 protein and 750 ng sgRNA for 1 h at 37 °C as previous study [ ]. .. For the positive control of the cleavage assay, same PCR products were cleaved in 1× NEBuffer 3.1 (100 mM NaCl, 50 mM Tris−HCl, 10 mM MgCl2 , 100 μg/ml BSA, New England BioLabs) condition. .. The RNase A (4 μg) was added to samples to remove sgRNA and the final samples were analyzed with agarose gel electrophoresis.

    Polymerase Chain Reaction:

    Article Title: CRISPR/dCas9-mediated biosensor for detection of tick-borne diseases
    Article Snippet: In 10 μl reactions, buffer mixed PCR products were incubated with 1 μg Cas9 protein and 750 ng sgRNA for 1 h at 37 °C as previous study [ ]. .. For the positive control of the cleavage assay, same PCR products were cleaved in 1× NEBuffer 3.1 (100 mM NaCl, 50 mM Tris−HCl, 10 mM MgCl2 , 100 μg/ml BSA, New England BioLabs) condition. .. The RNase A (4 μg) was added to samples to remove sgRNA and the final samples were analyzed with agarose gel electrophoresis.

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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.
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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

    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