nebuffer 3 1  (New England Biolabs)


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    Name:
    NEBuffer 3 1
    Description:
    NEBuffer 3 1 5 0 ml
    Catalog Number:
    B7203S
    Price:
    24
    Category:
    Buffers
    Size:
    5 0 ml
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    New England Biolabs nebuffer 3 1
    NEBuffer 3 1
    NEBuffer 3 1 5 0 ml
    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, 2021-09
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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 "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

    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 "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

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    Article Snippet: .. The nuclei were isolated using cell lysis buffer and resuspended in NEBuffer 3.1 (New England Biolabs Inc.). ..

    Lysis:

    Article Title: GATA3 maintains the quiescent state of cochlear supporting cells by regulating p27kip1
    Article Snippet: .. The nuclei were isolated using cell lysis buffer and resuspended in NEBuffer 3.1 (New England Biolabs Inc.). ..

    Hi-C:

    Article Title: A cohesin traffic pattern genetically linked to gene regulation
    Article Snippet: .. 5μg of Hi-C library were incubated with 5μL 10x NEBuffer 3.1, 0.025mM dATP, 0.025mM dGTP and 15U T4 DNA polymerase (NEB # M0203L) in 50µL. ..

    Incubation:

    Article Title: A cohesin traffic pattern genetically linked to gene regulation
    Article Snippet: .. 5μg of Hi-C library were incubated with 5μL 10x NEBuffer 3.1, 0.025mM dATP, 0.025mM dGTP and 15U T4 DNA polymerase (NEB # M0203L) in 50µL. ..

    Article Title: Biochemically distinct cohesin complexes mediate positioned loops between CTCF sites and dynamic loops within chromatin domains
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    New England Biolabs t7e1
    Myoediting strategy and identification of optimal guide RNAs to target the top 12 exons in DMD. ( A ) Conserved splice sites contain multiple NAG and NGG sequences, which enable cleavage by SpCas9. The numbers indicate the frequency of occurrence (%). ( B ) Human DMD exon structure. Shapes of intron-exon junctions indicate complementarity that maintains the open reading frame upon splicing. Red arrowheads indicate the top 12 targeted exons. The numbers indicate the order of the exons. ( C ) <t>T7E1</t> assays in human 293 cells transfected with plasmids expressing the corresponding guide RNA (gRNA), SpCas9, and GFP for the top 12 exons. The PCR products from GFP + and GFP − cells were cut with T7 endonuclease I (T7E1), which is specific to heteroduplex DNA caused by CRISPR/Cas9-mediated genome editing. Red arrowhead indicates cleavage bands of T7E1. bp indicates the base pair length of the marker bands.
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    New England Biolabs t4pol
    ExoCET mechanism. ( A ) Juxtaposition of the 80-bp homology arms between the p15A-cm (chloramphenicol) vector and the 14-kb lux genomic segment is illustrated: (a) both homology arms were located at the termini; (b and c) one homology arm was located at a terminus and the other 1 kb from the other end; (d) both homology arms were 1 kb from each end. ( B ) Number of colonies obtained from ETgA, <t>T4pol</t> or ExoCET using the homology arm combinations (a–d) as indicated. Reaction conditions were the same as for Figure 1F . ( C ) Protein combinations as indicated expressed from pSC101 plasmids in GB2005 were tested for direct cloning of the 14-kb lux gene cluster using terminal homology arms and ExoCET conditions except for the omission of RecA (ETg); RecA and RecT (Eg), RecA and RecE (Tg) and all (pSC101-tet). Error bars, s.d.; n = 3. Corresponding DNA analyses are shown in Supplementary Figure S5 .
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    New England Biolabs nebuffer 3
    Efficient gene deletion achieved following the treatment with CriPs targeting Gfp in primary GFP pre-adipocytes isolated from GFP mice. (A, C) Flow cytometry data of primary GFP pre-adipocytes treated with CriPs formulated in (A) <t>NEBuffer</t> 3 or (C) PBS. Cas9-sgRNA: 100 nM. (B, D) Different concentrations of EP with 100 nM of Cas9-sgRNA formulated in (B) NEBuffer 3 or (D) PBS. (E) Percent Indels measurements by T7E1 assay in genomic DNA isolated from primary GFP pre-adipocytes. Uncut: 401 bp, Cut: 288 bp + 113 bp.
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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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    Myoediting strategy and identification of optimal guide RNAs to target the top 12 exons in DMD. ( A ) Conserved splice sites contain multiple NAG and NGG sequences, which enable cleavage by SpCas9. The numbers indicate the frequency of occurrence (%). ( B ) Human DMD exon structure. Shapes of intron-exon junctions indicate complementarity that maintains the open reading frame upon splicing. Red arrowheads indicate the top 12 targeted exons. The numbers indicate the order of the exons. ( C ) T7E1 assays in human 293 cells transfected with plasmids expressing the corresponding guide RNA (gRNA), SpCas9, and GFP for the top 12 exons. The PCR products from GFP + and GFP − cells were cut with T7 endonuclease I (T7E1), which is specific to heteroduplex DNA caused by CRISPR/Cas9-mediated genome editing. Red arrowhead indicates cleavage bands of T7E1. bp indicates the base pair length of the marker bands.

    Journal: Science Advances

    Article Title: Correction of diverse muscular dystrophy mutations in human engineered heart muscle by single-site genome editing

    doi: 10.1126/sciadv.aap9004

    Figure Lengend Snippet: Myoediting strategy and identification of optimal guide RNAs to target the top 12 exons in DMD. ( A ) Conserved splice sites contain multiple NAG and NGG sequences, which enable cleavage by SpCas9. The numbers indicate the frequency of occurrence (%). ( B ) Human DMD exon structure. Shapes of intron-exon junctions indicate complementarity that maintains the open reading frame upon splicing. Red arrowheads indicate the top 12 targeted exons. The numbers indicate the order of the exons. ( C ) T7E1 assays in human 293 cells transfected with plasmids expressing the corresponding guide RNA (gRNA), SpCas9, and GFP for the top 12 exons. The PCR products from GFP + and GFP − cells were cut with T7 endonuclease I (T7E1), which is specific to heteroduplex DNA caused by CRISPR/Cas9-mediated genome editing. Red arrowhead indicates cleavage bands of T7E1. bp indicates the base pair length of the marker bands.

    Article Snippet: Following denaturation/renaturation, the following was added to the samples: 3 μl of 10× NEBuffer 2, 0.3 μl of T7E1 (New England Biolabs), and ddH2 O to 30 μl.

    Techniques: Transfection, Expressing, Polymerase Chain Reaction, CRISPR, Marker

    ExoCET mechanism. ( A ) Juxtaposition of the 80-bp homology arms between the p15A-cm (chloramphenicol) vector and the 14-kb lux genomic segment is illustrated: (a) both homology arms were located at the termini; (b and c) one homology arm was located at a terminus and the other 1 kb from the other end; (d) both homology arms were 1 kb from each end. ( B ) Number of colonies obtained from ETgA, T4pol or ExoCET using the homology arm combinations (a–d) as indicated. Reaction conditions were the same as for Figure 1F . ( C ) Protein combinations as indicated expressed from pSC101 plasmids in GB2005 were tested for direct cloning of the 14-kb lux gene cluster using terminal homology arms and ExoCET conditions except for the omission of RecA (ETg); RecA and RecT (Eg), RecA and RecE (Tg) and all (pSC101-tet). Error bars, s.d.; n = 3. Corresponding DNA analyses are shown in Supplementary Figure S5 .

    Journal: Nucleic Acids Research

    Article Title: ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes

    doi: 10.1093/nar/gkx1249

    Figure Lengend Snippet: ExoCET mechanism. ( A ) Juxtaposition of the 80-bp homology arms between the p15A-cm (chloramphenicol) vector and the 14-kb lux genomic segment is illustrated: (a) both homology arms were located at the termini; (b and c) one homology arm was located at a terminus and the other 1 kb from the other end; (d) both homology arms were 1 kb from each end. ( B ) Number of colonies obtained from ETgA, T4pol or ExoCET using the homology arm combinations (a–d) as indicated. Reaction conditions were the same as for Figure 1F . ( C ) Protein combinations as indicated expressed from pSC101 plasmids in GB2005 were tested for direct cloning of the 14-kb lux gene cluster using terminal homology arms and ExoCET conditions except for the omission of RecA (ETg); RecA and RecT (Eg), RecA and RecE (Tg) and all (pSC101-tet). Error bars, s.d.; n = 3. Corresponding DNA analyses are shown in Supplementary Figure S5 .

    Article Snippet: In vitro assembly Ten micrograms of genomic DNA and 200 ng of 2.2-kb p15A-cm linear vector (1 μg of 8-kb linear BAC vector) were assembled in 20 μl reactions consisting of 2 μl of 10 × NEBuffer 2.1 and 0.13 μl of 3 U μl−1 T4pol (NEB, cat. no. M0203).

    Techniques: Plasmid Preparation, Clone Assay

    Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic DNA and treated with 0.02 U μl −1 T4pol at 25°C for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).

    Journal: Nucleic Acids Research

    Article Title: ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes

    doi: 10.1093/nar/gkx1249

    Figure Lengend Snippet: Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic DNA and treated with 0.02 U μl −1 T4pol at 25°C for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).

    Article Snippet: In vitro assembly Ten micrograms of genomic DNA and 200 ng of 2.2-kb p15A-cm linear vector (1 μg of 8-kb linear BAC vector) were assembled in 20 μl reactions consisting of 2 μl of 10 × NEBuffer 2.1 and 0.13 μl of 3 U μl−1 T4pol (NEB, cat. no. M0203).

    Techniques: In Vitro, Clone Assay, Plasmid Preparation, Electroporation, Titration, Incubation, Standard Deviation

    Efficient gene deletion achieved following the treatment with CriPs targeting Gfp in primary GFP pre-adipocytes isolated from GFP mice. (A, C) Flow cytometry data of primary GFP pre-adipocytes treated with CriPs formulated in (A) NEBuffer 3 or (C) PBS. Cas9-sgRNA: 100 nM. (B, D) Different concentrations of EP with 100 nM of Cas9-sgRNA formulated in (B) NEBuffer 3 or (D) PBS. (E) Percent Indels measurements by T7E1 assay in genomic DNA isolated from primary GFP pre-adipocytes. Uncut: 401 bp, Cut: 288 bp + 113 bp.

    Journal: bioRxiv

    Article Title: CRISPR delivery particles for developing therapeutic strategies in metabolic disease

    doi: 10.1101/352799

    Figure Lengend Snippet: Efficient gene deletion achieved following the treatment with CriPs targeting Gfp in primary GFP pre-adipocytes isolated from GFP mice. (A, C) Flow cytometry data of primary GFP pre-adipocytes treated with CriPs formulated in (A) NEBuffer 3 or (C) PBS. Cas9-sgRNA: 100 nM. (B, D) Different concentrations of EP with 100 nM of Cas9-sgRNA formulated in (B) NEBuffer 3 or (D) PBS. (E) Percent Indels measurements by T7E1 assay in genomic DNA isolated from primary GFP pre-adipocytes. Uncut: 401 bp, Cut: 288 bp + 113 bp.

    Article Snippet: Cas9 protein and sgRNA were mixed in NEBuffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9) purchased from New England Biolabs Inc. or PBS at 37 °C for 10 minutes to form nano-size complexes.

    Techniques: Isolation, Mouse Assay, Flow Cytometry

    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