λ dna recombinant λ dna  (New England Biolabs)


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    Name:
    Lambda DNA
    Description:
    Lambda DNA 1 250 ug
    Catalog Number:
    n3011l
    Price:
    276
    Size:
    1 250 ug
    Category:
    Genomic DNA
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    New England Biolabs λ dna recombinant λ dna
    Lambda DNA
    Lambda DNA 1 250 ug
    https://www.bioz.com/result/λ dna recombinant λ dna/product/New England Biolabs
    Average 96 stars, based on 476 article reviews
    Price from $9.99 to $1999.99
    λ dna recombinant λ dna - by Bioz Stars, 2021-02
    96/100 stars

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    1) Product Images from "Efficient modification of λ-DNA substrates for single-molecule studies"

    Article Title: Efficient modification of λ-DNA substrates for single-molecule studies

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01984-x

    yRFC loads yPCNA on various extrahelical DNA structures. ( a ) Fluorescent images of yPCNA on DNA substrates with a 5′-ssDNA flap inserted at site A. Top: with buffer flow, bottom: without flow. DNA is stained with YOYO-1 (green) and yPCNA is labeled with anti-FLAG antibody conjugated QDs (magenta). Stopping buffer flow retracts both yPCNA and DNA to the Cr barrier, confirming that yPCNA is bound to the DNA and is not on the surface (bottom panel). ( b ) Binding distribution histogram of yPCNA/yRFC on λ-DNA molecules containing a 5′-ssDNA flap (top), (CAG) 13 repeat (middle), and homoduplex DNA (bottom). Red line: fit to a Gaussian distribution (center = 21.2 kb ± 1.4 kb; st. dev.). Pink box: a 5 kb window that captures 99% of all yPCNAs at site A (see text for details). ( c ) The mean number of yPCNA molecules loaded at site A (within the pink box), as determined by bootstrap analysis of the histograms in ( b ). Error bars represent the st. dev. of the mean from the bootstrap analysis.
    Figure Legend Snippet: yRFC loads yPCNA on various extrahelical DNA structures. ( a ) Fluorescent images of yPCNA on DNA substrates with a 5′-ssDNA flap inserted at site A. Top: with buffer flow, bottom: without flow. DNA is stained with YOYO-1 (green) and yPCNA is labeled with anti-FLAG antibody conjugated QDs (magenta). Stopping buffer flow retracts both yPCNA and DNA to the Cr barrier, confirming that yPCNA is bound to the DNA and is not on the surface (bottom panel). ( b ) Binding distribution histogram of yPCNA/yRFC on λ-DNA molecules containing a 5′-ssDNA flap (top), (CAG) 13 repeat (middle), and homoduplex DNA (bottom). Red line: fit to a Gaussian distribution (center = 21.2 kb ± 1.4 kb; st. dev.). Pink box: a 5 kb window that captures 99% of all yPCNAs at site A (see text for details). ( c ) The mean number of yPCNA molecules loaded at site A (within the pink box), as determined by bootstrap analysis of the histograms in ( b ). Error bars represent the st. dev. of the mean from the bootstrap analysis.

    Techniques Used: Flow Cytometry, Staining, Labeling, Binding Assay

    Single-molecule imaging of Lac repressor (LacI) dynamics on recombineered λ- DNA substrates. ( a ) Schematic of a single-tethered DNA curtain assay. DNA molecules (green) are immobilized on a fluid lipid bilayer and arranged at micro-fabricated Chromium (Cr) barriers. One of the insertion sites (site A) is indicated in blue. ( b ) TIRFM image of LacI (magenta) binding to a lac operator inserted at site A on a recombineered λ-DNA substrate (green). ( c ) Binding distributions of LacI to a lac operator sequence inserted at one of the three sites within λ-DNA. Solid lines indicate a Gaussian fit to the data with the indicated mean ± standard deviation. Blue: site A (21.7 ± 1.0 kb, N = 558, blue). Yellow: site B (34.0 ± 1.5 kb, mean N = 538). Orange: site C (46.0 ± 2.3 kb, N = 467). ( d ) Insertion of two lac operator cassettes within a single λ-DNA construct. Inset: single-molecule imaging of the recombinant DNA shows LacI at both positions B C. Binding histograms show nearly equal occupancy at both lac operator sites. Red line: double Gaussian fit (34.0 ± 2.0 kb and 46.2 ± 2.2 kb, respectively; N = 525). ( e ) As expected, LacI dissociates from both operator sites after injection of 0.4 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). ( f ) Quantification of the LacI lifetimes upon IPTG injection. The solid lines are single-exponential fits to the data (blue diamonds, t ½ = 21 ± 1.0 sec, N = 63, and orange squares, t ½ = 22 ± 0.8 sec, N = 56 at positions B C, respectfully). The solid gray line is the single-exponential fit of the LacI lifetime data, in the absence of IPTG (grey circles, t ½ = 327 ± 10 s, N = 49). The error bars indicate the standard deviation obtained via bootstrap analysis.
    Figure Legend Snippet: Single-molecule imaging of Lac repressor (LacI) dynamics on recombineered λ- DNA substrates. ( a ) Schematic of a single-tethered DNA curtain assay. DNA molecules (green) are immobilized on a fluid lipid bilayer and arranged at micro-fabricated Chromium (Cr) barriers. One of the insertion sites (site A) is indicated in blue. ( b ) TIRFM image of LacI (magenta) binding to a lac operator inserted at site A on a recombineered λ-DNA substrate (green). ( c ) Binding distributions of LacI to a lac operator sequence inserted at one of the three sites within λ-DNA. Solid lines indicate a Gaussian fit to the data with the indicated mean ± standard deviation. Blue: site A (21.7 ± 1.0 kb, N = 558, blue). Yellow: site B (34.0 ± 1.5 kb, mean N = 538). Orange: site C (46.0 ± 2.3 kb, N = 467). ( d ) Insertion of two lac operator cassettes within a single λ-DNA construct. Inset: single-molecule imaging of the recombinant DNA shows LacI at both positions B C. Binding histograms show nearly equal occupancy at both lac operator sites. Red line: double Gaussian fit (34.0 ± 2.0 kb and 46.2 ± 2.2 kb, respectively; N = 525). ( e ) As expected, LacI dissociates from both operator sites after injection of 0.4 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). ( f ) Quantification of the LacI lifetimes upon IPTG injection. The solid lines are single-exponential fits to the data (blue diamonds, t ½ = 21 ± 1.0 sec, N = 63, and orange squares, t ½ = 22 ± 0.8 sec, N = 56 at positions B C, respectfully). The solid gray line is the single-exponential fit of the LacI lifetime data, in the absence of IPTG (grey circles, t ½ = 327 ± 10 s, N = 49). The error bars indicate the standard deviation obtained via bootstrap analysis.

    Techniques Used: Imaging, Binding Assay, Sequencing, Standard Deviation, Construct, Recombinant, Injection, Size-exclusion Chromatography

    Recombineering at three unique positions within the λ-phage genome. ( a ) Insertion cassettes were designed for three sites targeting dispensable segments of the λ-phage genome (shown in gray). These cassettes are 21.3 kb, 33.5 kb and 45.3 kb away from cosL (designated A, B and C, respectively). ( b ) Schematic of the Red-based recombineering. An arabinose-inducible plasmid supplies the three Red genes. ( c ) Cassettes for efficient recombineering into each of the sites shown in ( a ). The cassettes carry an antibiotic resistance gene that is flanked on both sides by ~200 bp of homology to λ-DNA. ( d ) Recombineering efficiency was scored by colony PCR followed by agarose gels analysis. Successful recombineering generates a smaller PCR fragment. We observed 90–100% insertion efficiency at each of the three target sites.
    Figure Legend Snippet: Recombineering at three unique positions within the λ-phage genome. ( a ) Insertion cassettes were designed for three sites targeting dispensable segments of the λ-phage genome (shown in gray). These cassettes are 21.3 kb, 33.5 kb and 45.3 kb away from cosL (designated A, B and C, respectively). ( b ) Schematic of the Red-based recombineering. An arabinose-inducible plasmid supplies the three Red genes. ( c ) Cassettes for efficient recombineering into each of the sites shown in ( a ). The cassettes carry an antibiotic resistance gene that is flanked on both sides by ~200 bp of homology to λ-DNA. ( d ) Recombineering efficiency was scored by colony PCR followed by agarose gels analysis. Successful recombineering generates a smaller PCR fragment. We observed 90–100% insertion efficiency at each of the three target sites.

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction

    yPCNA diffusion on λ-DNA containing various extrahelical structures. ( a ) An illustration of the double-tethered DNA curtains. Chromium (Cr) pedestals are coated with anti-digoxigenin antibodies, and buffer flow is used to immobilize the dig-labeled λ-DNA between the linear barriers and Cr pedestals. Kymograph of diffusing yPCNA molecule on DNA substrates with ( b ) homoduplex DNA ( c ) a 5′-ssDNA flap or a (CAG) 13 repeat ( d ). The characteristic changes in diffusion behavior of yPCNA are indicated with arrows, and the dashed lines indicate site A. ( e ) Percentage of molecules showing either bypass, blocked, or captured behavior at site A. At least 35 DNA molecules were analyzed and classified into each of three categories (N = 40, 36, and 40 for the flap, (CAG) 13 , and homoduplex DNA substrates).
    Figure Legend Snippet: yPCNA diffusion on λ-DNA containing various extrahelical structures. ( a ) An illustration of the double-tethered DNA curtains. Chromium (Cr) pedestals are coated with anti-digoxigenin antibodies, and buffer flow is used to immobilize the dig-labeled λ-DNA between the linear barriers and Cr pedestals. Kymograph of diffusing yPCNA molecule on DNA substrates with ( b ) homoduplex DNA ( c ) a 5′-ssDNA flap or a (CAG) 13 repeat ( d ). The characteristic changes in diffusion behavior of yPCNA are indicated with arrows, and the dashed lines indicate site A. ( e ) Percentage of molecules showing either bypass, blocked, or captured behavior at site A. At least 35 DNA molecules were analyzed and classified into each of three categories (N = 40, 36, and 40 for the flap, (CAG) 13 , and homoduplex DNA substrates).

    Techniques Used: Diffusion-based Assay, Flow Cytometry, Labeling

    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 "Inserting Extrahelical Structures into Long DNA Substrates for Single-Molecule Studies of DNA Mismatch Repair"

    Article Title: Inserting Extrahelical Structures into Long DNA Substrates for Single-Molecule Studies of DNA Mismatch Repair

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2016.08.006

    A strategy for inserting extrahelical structures into λ-DNA. Step 1: A nicking cassette is inserted into the λ-phage genome in vivo . Step 2: Recombinant λ-DNA is purified. Step 3: Extrahelical structures are introduced via a nicking enzyme-based oligonucleotide insertion strategy. B and D represent incorporated biotinylated and digoxigenin-labeled oligonucleotides, respectively. Step 4: The resulting DNA substrates are assembled into microfluidic DNA curtains and imaged via single-molecule microscopy.
    Figure Legend Snippet: A strategy for inserting extrahelical structures into λ-DNA. Step 1: A nicking cassette is inserted into the λ-phage genome in vivo . Step 2: Recombinant λ-DNA is purified. Step 3: Extrahelical structures are introduced via a nicking enzyme-based oligonucleotide insertion strategy. B and D represent incorporated biotinylated and digoxigenin-labeled oligonucleotides, respectively. Step 4: The resulting DNA substrates are assembled into microfluidic DNA curtains and imaged via single-molecule microscopy.

    Techniques Used: In Vivo, Recombinant, Purification, Labeling, Microscopy

    Constructing λ-DNA with an internal single-stranded DNA flap. (a) Schematic of the nickase-based oligonucleotide replacement reaction. B and D represent biotinylated and digoxigenin-labeled oligonucleotides, respectively. (b) A restriction digest can be used to quantify oligonucleotide replacement rapidly. Inserting a 5′-ssDNA flap, but not a homoduplex oligonucleotide, produces a 2.7-kb fragment. Homoduplex and mock-treated λ-DNA are further digested into 2 and 0.7-kb fragments (0.7-kb band not shown). (c) A denaturing (alkaline) agarose gel confirms insertion and religation of the λ-DNA substrates. Note that the top and bottom DNA strands are separated only for the 5′-ssDNA flap substrate.
    Figure Legend Snippet: Constructing λ-DNA with an internal single-stranded DNA flap. (a) Schematic of the nickase-based oligonucleotide replacement reaction. B and D represent biotinylated and digoxigenin-labeled oligonucleotides, respectively. (b) A restriction digest can be used to quantify oligonucleotide replacement rapidly. Inserting a 5′-ssDNA flap, but not a homoduplex oligonucleotide, produces a 2.7-kb fragment. Homoduplex and mock-treated λ-DNA are further digested into 2 and 0.7-kb fragments (0.7-kb band not shown). (c) A denaturing (alkaline) agarose gel confirms insertion and religation of the λ-DNA substrates. Note that the top and bottom DNA strands are separated only for the 5′-ssDNA flap substrate.

    Techniques Used: Labeling, Agarose Gel Electrophoresis

    Visualizing Msh2–Msh3 binding to an extrahelical ssDNA flap. (A) Cartoon representation ( left ) and picture ( right ) of a microscope-mounted flowcell. The picture highlights the microfluidic connectors and the quartz prism. (B) Distribution of Msh2–Msh3 molecules on flap-containing λ-DNA. The black line is a Gaussian fit to the data ( n =503). The center of the peak corresponds to the expected location of the lesion (20 kb from the top DNA barrier, error is SD). Inset : Seven representative λ-DNA molecules ( light gray vertical lines ) with flap-bound Msh2–Msh3 ( black points ). Msh2–Msh3 recognizes lesions via (C) 1D diffusion along the DNA or (D) a direct encounter (3D collision). Each panel shows a cartoon illustration ( top ), kymograph ( middle ), and single-particle trajectory of Msh2–Msh3 ( black ) binding a DNA flap (3′-ssDNA flap; marked as 3′). Asterisk indicates transient binding by a second Msh2–Msh3. In both cases, Msh2–Msh3 releases the flap and continues to diffuse on homoduplex λ-DNA.
    Figure Legend Snippet: Visualizing Msh2–Msh3 binding to an extrahelical ssDNA flap. (A) Cartoon representation ( left ) and picture ( right ) of a microscope-mounted flowcell. The picture highlights the microfluidic connectors and the quartz prism. (B) Distribution of Msh2–Msh3 molecules on flap-containing λ-DNA. The black line is a Gaussian fit to the data ( n =503). The center of the peak corresponds to the expected location of the lesion (20 kb from the top DNA barrier, error is SD). Inset : Seven representative λ-DNA molecules ( light gray vertical lines ) with flap-bound Msh2–Msh3 ( black points ). Msh2–Msh3 recognizes lesions via (C) 1D diffusion along the DNA or (D) a direct encounter (3D collision). Each panel shows a cartoon illustration ( top ), kymograph ( middle ), and single-particle trajectory of Msh2–Msh3 ( black ) binding a DNA flap (3′-ssDNA flap; marked as 3′). Asterisk indicates transient binding by a second Msh2–Msh3. In both cases, Msh2–Msh3 releases the flap and continues to diffuse on homoduplex λ-DNA.

    Techniques Used: Binding Assay, Microscopy, Diffusion-based Assay

    4) Product Images from "Antibody discovery and engineering by enhanced CRISPR-Cas9 integration of variable gene cassette libraries in mammalian cells"

    Article Title: Antibody discovery and engineering by enhanced CRISPR-Cas9 integration of variable gene cassette libraries in mammalian cells

    Journal: mAbs

    doi: 10.1080/19420862.2019.1662691

    Enhanced Cas9-driven HDR by designing a self-linearized donor plasmid. (a) Schematic shows the PnP workflow to reprogram mRuby hybridomas to express a selected antibody following incorporation of a recombinant synthetic antibody in the V H locus. 27 (b) Optimized workflow in which a version of the PnP-mRuby cells constitutively expressing Cas9 26 is used together with an HDR donor plasmid harboring a recognition site for the same Cas9 gRNA that is used to cleave mRuby (protospacer adjacent motif is indicated in red). Following entry of the plasmid and the gRNA complex into the nucleus, Cas9, which is also targeted to the nucleus due to its nuclear localization signal, is recruited to both induce a DSB in the genomic mRuby coding sequence and linearize the plasmid, rendering it more prone to integration by HDR. (c) In vitro testing of plasmid cleavage by recombinant Cas9. Three versions of the self-linearizing plasmid were generated, bearing the cleavage site upstream of the 5ʹ homology arm (pPnP-lin5ʹ), downstream of the 3ʹ homology arm (pPnP-lin3ʹ) and at both sites (pPnP-lin5ʹ/3ʹ). As expected, in the first two cases Cas9 cleavage produced a linearized construct, while the double-cut plasmid underwent both single cleavage at either site or simultaneous cleavage at both, resulting in a shorter construct. (d) Exemplary flow cytometry dot plots show evaluation of HDR integration. The improved plasmids were compared to the unmodified plasmid (pPnP) and to the PCR-linearized donor (PCR). HDR efficiency is evaluated in terms of surface antibody expression 3 d after transfection. (e) Fold improvement of HDR rates of all the linearized donor formats compared to the mean of unmodified plasmid. HDR efficiency is quantified as described in (d). The plot is representative of n = 3 replicates and the error bars indicate standard deviation. Flow cytometry dot plots of all replicates are shown in Figure S1.
    Figure Legend Snippet: Enhanced Cas9-driven HDR by designing a self-linearized donor plasmid. (a) Schematic shows the PnP workflow to reprogram mRuby hybridomas to express a selected antibody following incorporation of a recombinant synthetic antibody in the V H locus. 27 (b) Optimized workflow in which a version of the PnP-mRuby cells constitutively expressing Cas9 26 is used together with an HDR donor plasmid harboring a recognition site for the same Cas9 gRNA that is used to cleave mRuby (protospacer adjacent motif is indicated in red). Following entry of the plasmid and the gRNA complex into the nucleus, Cas9, which is also targeted to the nucleus due to its nuclear localization signal, is recruited to both induce a DSB in the genomic mRuby coding sequence and linearize the plasmid, rendering it more prone to integration by HDR. (c) In vitro testing of plasmid cleavage by recombinant Cas9. Three versions of the self-linearizing plasmid were generated, bearing the cleavage site upstream of the 5ʹ homology arm (pPnP-lin5ʹ), downstream of the 3ʹ homology arm (pPnP-lin3ʹ) and at both sites (pPnP-lin5ʹ/3ʹ). As expected, in the first two cases Cas9 cleavage produced a linearized construct, while the double-cut plasmid underwent both single cleavage at either site or simultaneous cleavage at both, resulting in a shorter construct. (d) Exemplary flow cytometry dot plots show evaluation of HDR integration. The improved plasmids were compared to the unmodified plasmid (pPnP) and to the PCR-linearized donor (PCR). HDR efficiency is evaluated in terms of surface antibody expression 3 d after transfection. (e) Fold improvement of HDR rates of all the linearized donor formats compared to the mean of unmodified plasmid. HDR efficiency is quantified as described in (d). The plot is representative of n = 3 replicates and the error bars indicate standard deviation. Flow cytometry dot plots of all replicates are shown in Figure S1.

    Techniques Used: Plasmid Preparation, Recombinant, Expressing, Sequencing, In Vitro, Generated, Produced, Construct, Flow Cytometry, Cytometry, Polymerase Chain Reaction, Transfection, Standard Deviation

    Characterization of the antibody discovered from immune library screening. (a) Lineage tree of the 41 unique clones retrieved after single-cell sorting. Somatic hypermutations (SHM) refer to non-silent amino acid changes to the V-germline gene. The five clones highlighted in blue were selected as representative due to sequence diversity. V- and J-germline gene usage, CDRH3 sequences and amino acid changes were retrieved via IMGT/V-quest ( http://www.imgt.org/IMGT_vquest/vquest ). (b) Supernatant ELISA of the five clones highlighted in (a) and whose surface expression profile is shown in Figure 2c . Two technical replicates were included for each sample and a five-parameter logistical curve was fitted to the data by nonlinear regression. For each data point, the mean is represented and the error bars indicate standard deviation. PnP-mRuby-Cas9 cell supernatant was used as negative control.
    Figure Legend Snippet: Characterization of the antibody discovered from immune library screening. (a) Lineage tree of the 41 unique clones retrieved after single-cell sorting. Somatic hypermutations (SHM) refer to non-silent amino acid changes to the V-germline gene. The five clones highlighted in blue were selected as representative due to sequence diversity. V- and J-germline gene usage, CDRH3 sequences and amino acid changes were retrieved via IMGT/V-quest ( http://www.imgt.org/IMGT_vquest/vquest ). (b) Supernatant ELISA of the five clones highlighted in (a) and whose surface expression profile is shown in Figure 2c . Two technical replicates were included for each sample and a five-parameter logistical curve was fitted to the data by nonlinear regression. For each data point, the mean is represented and the error bars indicate standard deviation. PnP-mRuby-Cas9 cell supernatant was used as negative control.

    Techniques Used: Library Screening, Clone Assay, FACS, Sequencing, Enzyme-linked Immunosorbent Assay, Expressing, Standard Deviation, Negative Control

    Characterization of antibody variants selected for affinity maturation. (a) The table shows the coding mutations of each of the five unique clones (HEL23v1-5) retrieved after single-cell sorting of the antigen-enriched libraries. (b) Flow cytometry dot plots show the surface expression and HEL-binding profile of the five HEL23 variants (green) and wild-type HEL23 (black). The ratio of HEL over antibody signal for each clone is shown in Figure S5a. (c) Supernatant ELISA comparing HEL binding for the five isolated variants (green) with wild-type HEL23 (black). Supernatants were adjusted to equal IgG-concentration (Fig. S5b). Two technical replicates were included for each of the mutated variants and one for the controls (PnP-HEL23 and PnP-mRuby-Cas9), and a five-parameter logistical curve was fitted to the data by nonlinear regression. For each data point, the mean is represented and the error bars indicate standard deviation. PnP-mRuby-Cas9 cell supernatant was used as negative control. (d) Affinity values of wild-type HEL23 and the five isolated variants obtained by bio-layer interferometry (BLI). The curves and fitting values are reported in Figure S6. (e) Heatmap shows enrichment of all possible substitutions for each WT residue (x-axis) in the HEL + library compared to IgG + library in a log2-fold change scale. Red substitutions are enriched in the HEL + library, while blue substitutions are depleted. White squares (log2 ratio = 0) indicate neutral substitutions not impacted by enrichment, or residues in which the mutation rate is negligible. The mutation rate for each position was calculated by summing the mutation frequency of all replicates (n = 3) for each condition. Key residues found in affinity-matured clones are indicated with black boxes.
    Figure Legend Snippet: Characterization of antibody variants selected for affinity maturation. (a) The table shows the coding mutations of each of the five unique clones (HEL23v1-5) retrieved after single-cell sorting of the antigen-enriched libraries. (b) Flow cytometry dot plots show the surface expression and HEL-binding profile of the five HEL23 variants (green) and wild-type HEL23 (black). The ratio of HEL over antibody signal for each clone is shown in Figure S5a. (c) Supernatant ELISA comparing HEL binding for the five isolated variants (green) with wild-type HEL23 (black). Supernatants were adjusted to equal IgG-concentration (Fig. S5b). Two technical replicates were included for each of the mutated variants and one for the controls (PnP-HEL23 and PnP-mRuby-Cas9), and a five-parameter logistical curve was fitted to the data by nonlinear regression. For each data point, the mean is represented and the error bars indicate standard deviation. PnP-mRuby-Cas9 cell supernatant was used as negative control. (d) Affinity values of wild-type HEL23 and the five isolated variants obtained by bio-layer interferometry (BLI). The curves and fitting values are reported in Figure S6. (e) Heatmap shows enrichment of all possible substitutions for each WT residue (x-axis) in the HEL + library compared to IgG + library in a log2-fold change scale. Red substitutions are enriched in the HEL + library, while blue substitutions are depleted. White squares (log2 ratio = 0) indicate neutral substitutions not impacted by enrichment, or residues in which the mutation rate is negligible. The mutation rate for each position was calculated by summing the mutation frequency of all replicates (n = 3) for each condition. Key residues found in affinity-matured clones are indicated with black boxes.

    Techniques Used: Clone Assay, FACS, Flow Cytometry, Cytometry, Expressing, Binding Assay, Enzyme-linked Immunosorbent Assay, Isolation, Concentration Assay, Standard Deviation, Negative Control, Mutagenesis

    5) 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

    6) 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

    7) 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

    8) Product Images from "Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection"

    Article Title: Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection

    Journal: Journal of Virology

    doi: 10.1128/JVI.00539-17

    Exo I III versus Exo T5 digestion of plasmid DNA. (A) Diagrams showing expected digestion results of various plasmid DNA species. A break in the circle denotes the nick on the DNA strand. (B and C) Plasmid pCI-HBc (2.5 ng) was mixed with 20 μl of mock PF DNA extracted from uninduced HepAD38 cells. The DNA mix was first treated with Nb.BbvCI (5 units) to nick the plasmid DNA specifically on the minus strand (B and C, lanes 5 to 8) or was left untreated (B and C, lanes 1 to 4) before digestion with Exo I III (5 units and 25 units, respectively) in two different buffers or with Exo T5 (5 units). The DNA samples were then resolved on an agarose gel, and HBc DNA was detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. B3, 1× NEB buffer 3; BCS, 1× NEB buffer Cutsmart; PE, phenol extraction.
    Figure Legend Snippet: Exo I III versus Exo T5 digestion of plasmid DNA. (A) Diagrams showing expected digestion results of various plasmid DNA species. A break in the circle denotes the nick on the DNA strand. (B and C) Plasmid pCI-HBc (2.5 ng) was mixed with 20 μl of mock PF DNA extracted from uninduced HepAD38 cells. The DNA mix was first treated with Nb.BbvCI (5 units) to nick the plasmid DNA specifically on the minus strand (B and C, lanes 5 to 8) or was left untreated (B and C, lanes 1 to 4) before digestion with Exo I III (5 units and 25 units, respectively) in two different buffers or with Exo T5 (5 units). The DNA samples were then resolved on an agarose gel, and HBc DNA was detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. B3, 1× NEB buffer 3; BCS, 1× NEB buffer Cutsmart; PE, phenol extraction.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Southern Blot, Migration

    Confirmation of the closed circular minus strand in the processed RC DNA by BmgBI or Nt.BbvCI and Exo I III digestion. (A and D) Diagrams showing expected results of digestion performed with various HBV PF DNA species. The short line intersecting the circle denotes the site of BmgBI digestion (A) or Nt.BbvCI nicking (D). The presence of the RNA (short gray line) at the 5′ end of the plus strand in RC DNA prevents BmgBI digestion (panel A; arrow blocked by a short line). The black dot at the 5′ end of the minus strand of the PF-RC DNA denotes the unknown modification of this end upon removal of the RT protein. The DNA species indicated in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in this study (see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (lanes 1 to 3) or PF DNA (lanes 4 to 6) extracted from induced HepAD38 cells was treated with BmgBI (5 units) in 1× NEB buffer 3 to linearize all supercoiled and nicked CCC DNA (lanes 2, 3, 5, and 6) or was mock treated (lanes 1 and 4). For lanes 3 and 6, the DNA samples were further digested with Exo I III after BmgBI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. (E) PF DNA extracted from induced HepAD38 cells was treated with Nt.BbvCI (5 units) in 1× NEB Cutsmart buffer to nick all CCC DNA (lanes 3, 4, 7, and 8) or mock treated (lanes 1 and 5). For lanes 4 and 8, the DNA samples were further digested with Exo I III after Nt.BbvCI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (lanes 1 to 4) or minus-strand (lanes 5 to 8) DNA. The diagrams on the right depict the various DNA species and their migration on the gel. Marker, the DNA marker lane. The size of the DNA markers is indicated (in kilobase pairs). The blank spaces between the lanes in panels B, C, and E indicate where other lanes from the same gel that were deemed nonessential for this work were cropped out during the preparation of the figure.
    Figure Legend Snippet: Confirmation of the closed circular minus strand in the processed RC DNA by BmgBI or Nt.BbvCI and Exo I III digestion. (A and D) Diagrams showing expected results of digestion performed with various HBV PF DNA species. The short line intersecting the circle denotes the site of BmgBI digestion (A) or Nt.BbvCI nicking (D). The presence of the RNA (short gray line) at the 5′ end of the plus strand in RC DNA prevents BmgBI digestion (panel A; arrow blocked by a short line). The black dot at the 5′ end of the minus strand of the PF-RC DNA denotes the unknown modification of this end upon removal of the RT protein. The DNA species indicated in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in this study (see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (lanes 1 to 3) or PF DNA (lanes 4 to 6) extracted from induced HepAD38 cells was treated with BmgBI (5 units) in 1× NEB buffer 3 to linearize all supercoiled and nicked CCC DNA (lanes 2, 3, 5, and 6) or was mock treated (lanes 1 and 4). For lanes 3 and 6, the DNA samples were further digested with Exo I III after BmgBI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. (E) PF DNA extracted from induced HepAD38 cells was treated with Nt.BbvCI (5 units) in 1× NEB Cutsmart buffer to nick all CCC DNA (lanes 3, 4, 7, and 8) or mock treated (lanes 1 and 5). For lanes 4 and 8, the DNA samples were further digested with Exo I III after Nt.BbvCI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (lanes 1 to 4) or minus-strand (lanes 5 to 8) DNA. The diagrams on the right depict the various DNA species and their migration on the gel. Marker, the DNA marker lane. The size of the DNA markers is indicated (in kilobase pairs). The blank spaces between the lanes in panels B, C, and E indicate where other lanes from the same gel that were deemed nonessential for this work were cropped out during the preparation of the figure.

    Techniques Used: Modification, Countercurrent Chromatography, Agarose Gel Electrophoresis, Southern Blot, Migration, Marker

    9) Product Images from "A point mutation decouples the lipid transfer activities of microsomal triglyceride transfer protein"

    Article Title: A point mutation decouples the lipid transfer activities of microsomal triglyceride transfer protein

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008941

    The stl and c655 mttp mutations have differential effects on growth and the accumulation of lipid in intestine and liver. (A) Representative images of male WT and mttp mutant fish at 12 weeks of age. (B) Representative images of H E stained intestine and liver from adult male WT and mttp mutant fish (7.5 mo), scale = 50 μm, * indicate goblet cells, arrows indicate representative lipid accumulation in enterocytes. (C–E) Intestine and liver tissue from adult male fish were extracted based on equal concentration of protein. Tissue lipid extracts from WT and mttp mutant fish were quantitated using an HPLC system coupled to a tandem mass spectrometer (LC-MS/MS) (n = 3; 1 fish per sample/genotype). (C) Heat maps represent fold-change from WT of over 1000 individual lipid species grouped into lipid classes (triacylglycerol [TG, n = 274], diacylglycerol [DG, n = 108], mo noacylglycerol [MG, n = 36], sphingomyelin [SM, n = 72], cholesterol ester [CE, n = 7], ceramides [Cer, n = 44], phospholipid [PL, n = 472], free fatty acid [FA, n = 27] and other lipids [O; including sterols, sphingosine, sulfatide, zymosteryl and wax esters, n = 10]). (D) Quantification of total intestinal and liver TG, DG, PL, and FA from mutant lines as expressed as a sum of lipid group (n = 3). For additional lipid groups, see S11 Fig . (E) The number of individual lipid species data from panel (C) that are statistically different from WT (adj. p
    Figure Legend Snippet: The stl and c655 mttp mutations have differential effects on growth and the accumulation of lipid in intestine and liver. (A) Representative images of male WT and mttp mutant fish at 12 weeks of age. (B) Representative images of H E stained intestine and liver from adult male WT and mttp mutant fish (7.5 mo), scale = 50 μm, * indicate goblet cells, arrows indicate representative lipid accumulation in enterocytes. (C–E) Intestine and liver tissue from adult male fish were extracted based on equal concentration of protein. Tissue lipid extracts from WT and mttp mutant fish were quantitated using an HPLC system coupled to a tandem mass spectrometer (LC-MS/MS) (n = 3; 1 fish per sample/genotype). (C) Heat maps represent fold-change from WT of over 1000 individual lipid species grouped into lipid classes (triacylglycerol [TG, n = 274], diacylglycerol [DG, n = 108], mo noacylglycerol [MG, n = 36], sphingomyelin [SM, n = 72], cholesterol ester [CE, n = 7], ceramides [Cer, n = 44], phospholipid [PL, n = 472], free fatty acid [FA, n = 27] and other lipids [O; including sterols, sphingosine, sulfatide, zymosteryl and wax esters, n = 10]). (D) Quantification of total intestinal and liver TG, DG, PL, and FA from mutant lines as expressed as a sum of lipid group (n = 3). For additional lipid groups, see S11 Fig . (E) The number of individual lipid species data from panel (C) that are statistically different from WT (adj. p

    Techniques Used: Mutagenesis, Fluorescence In Situ Hybridization, Staining, Concentration Assay, High Performance Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

    Structural analysis of MTP mutations. (A) Ribbon representation of the human MTP complex (PDB entry 6I7S) and the Zebrafish modeled structure. The positions of L475 and G863 in the Zebrafish structure are shown in space-filling representation. (B) Alignment of human MTP and zebrafish Mtp amino acid sequences surrounding the stl and c655 mutations. (C) Close-up view of the area surrounding L477 in the human MTP complex. The position of L477 (red) is highlighted. The conserved hydrogen bonds linking the helical domain to the tip of the C-sheet of the lipid-binding domain are shown as well as amino acids within 4Å of L477. (D) Close-up view of the area surrounding G865 in the human MTP complex. The position of G865 (yellow) and the PEG molecule (dark green) which occupies the lipid-binding site in the solved structure are shown in space-filling representation. The a’ domain of PDI (pink) in the complex occludes the lipid entry/exit site. (E) Close-up view showing the outer strand displacement in sheet A of the lipid-binding domain of the M subunit resulting from the G865V mutation. Asterisk indicates the wild-type backbone carbonyl of G865 hydrogen bonded to R461 of PDI. Panels C–E are colored as in panel A.
    Figure Legend Snippet: Structural analysis of MTP mutations. (A) Ribbon representation of the human MTP complex (PDB entry 6I7S) and the Zebrafish modeled structure. The positions of L475 and G863 in the Zebrafish structure are shown in space-filling representation. (B) Alignment of human MTP and zebrafish Mtp amino acid sequences surrounding the stl and c655 mutations. (C) Close-up view of the area surrounding L477 in the human MTP complex. The position of L477 (red) is highlighted. The conserved hydrogen bonds linking the helical domain to the tip of the C-sheet of the lipid-binding domain are shown as well as amino acids within 4Å of L477. (D) Close-up view of the area surrounding G865 in the human MTP complex. The position of G865 (yellow) and the PEG molecule (dark green) which occupies the lipid-binding site in the solved structure are shown in space-filling representation. The a’ domain of PDI (pink) in the complex occludes the lipid entry/exit site. (E) Close-up view showing the outer strand displacement in sheet A of the lipid-binding domain of the M subunit resulting from the G865V mutation. Asterisk indicates the wild-type backbone carbonyl of G865 hydrogen bonded to R461 of PDI. Panels C–E are colored as in panel A.

    Techniques Used: Binding Assay, Mutagenesis

    The c655 allele is a missense mutation in the M-subunit of microsomal triglyceride transfer protein. (A) Representative images of a wild-type zebrafish embryo, a homozygous mutant embryo carrying the previously described stalactite ( stl ) missense mutation in mttp , and a homozygous c655 mutant embryo at 3 days post fertilization (dpf); Scale = 500 μm. The dark/opaque yolk phenotype in embryos from c655 heterozygous in-crosses segregated with a Mendelian ratio consistent with a homozygous recessive mutation, mean +/- SD. For source data, see S4 File . (B) Euclidean distance mapping analysis plots produced by MMAPPR [ 51 ], showing the likely genomic region of the c655 mutation. Plot of the LOESS fit to the mapping scores (Euclidean Distance 4 ) across all 25 chromosomes (top) and expanded view of chromosome 1(GRCz10: CM002885.1) (bottom). Single nucleotide variants (SNVs) present in this 11 MB region in c655 mutant embryos were assessed for their effect on annotated genes using the Ensembl Variant Effect Predictor [ 52 ], including using the Sorting Intolerant from Tolerant algorithm (SIFT) [ 53 ], to predict the impact of changes on protein-coding sequence (tolerated or deleterious). We extracted variants that alter the protein-coding sequence as candidates for the causal mutation (223 variants in 64 genes, of which 42 are missense variants predicted to be deleterious; S1 File ). One of the SNVs linked to the c655 phenotype was a G > T missense mutation predicted to be deleterious in exon 18 of the microsomal triglyceride transfer protein gene (ENSDARG00000008637, Chr1:11,421,261 GRCz10, red arrow in B shows the position of the G > T missense mutation in mttp ). (C) Representative image of a trans-heterozygous mttp stl/c655 embryo; 3 dpf, scale = 500 μm. The dark/opaque yolk phenotype is present at expected ratios and genotyping confirms that only the embryos with opaque yolks are trans-heterozygous for the mttp alleles. (D) Depiction of the mttp gene structure highlighting the locations of the stl (L475P) (position 11431645 (GRCz10), transcript mtp-204 (ENSDART00000165753.2)) and c655 (G863V) missense alleles in exon 11 and 18, respectively. An additional SNV in mttp at position Chr1:11,421,300 GRCz10 (T > C) causing a missense mutation (M850T) was also identified in c655 mutants; however, this SNV was not predicted to be deleterious and has been previously noted in Ensembl.
    Figure Legend Snippet: The c655 allele is a missense mutation in the M-subunit of microsomal triglyceride transfer protein. (A) Representative images of a wild-type zebrafish embryo, a homozygous mutant embryo carrying the previously described stalactite ( stl ) missense mutation in mttp , and a homozygous c655 mutant embryo at 3 days post fertilization (dpf); Scale = 500 μm. The dark/opaque yolk phenotype in embryos from c655 heterozygous in-crosses segregated with a Mendelian ratio consistent with a homozygous recessive mutation, mean +/- SD. For source data, see S4 File . (B) Euclidean distance mapping analysis plots produced by MMAPPR [ 51 ], showing the likely genomic region of the c655 mutation. Plot of the LOESS fit to the mapping scores (Euclidean Distance 4 ) across all 25 chromosomes (top) and expanded view of chromosome 1(GRCz10: CM002885.1) (bottom). Single nucleotide variants (SNVs) present in this 11 MB region in c655 mutant embryos were assessed for their effect on annotated genes using the Ensembl Variant Effect Predictor [ 52 ], including using the Sorting Intolerant from Tolerant algorithm (SIFT) [ 53 ], to predict the impact of changes on protein-coding sequence (tolerated or deleterious). We extracted variants that alter the protein-coding sequence as candidates for the causal mutation (223 variants in 64 genes, of which 42 are missense variants predicted to be deleterious; S1 File ). One of the SNVs linked to the c655 phenotype was a G > T missense mutation predicted to be deleterious in exon 18 of the microsomal triglyceride transfer protein gene (ENSDARG00000008637, Chr1:11,421,261 GRCz10, red arrow in B shows the position of the G > T missense mutation in mttp ). (C) Representative image of a trans-heterozygous mttp stl/c655 embryo; 3 dpf, scale = 500 μm. The dark/opaque yolk phenotype is present at expected ratios and genotyping confirms that only the embryos with opaque yolks are trans-heterozygous for the mttp alleles. (D) Depiction of the mttp gene structure highlighting the locations of the stl (L475P) (position 11431645 (GRCz10), transcript mtp-204 (ENSDART00000165753.2)) and c655 (G863V) missense alleles in exon 11 and 18, respectively. An additional SNV in mttp at position Chr1:11,421,300 GRCz10 (T > C) causing a missense mutation (M850T) was also identified in c655 mutants; however, this SNV was not predicted to be deleterious and has been previously noted in Ensembl.

    Techniques Used: Mutagenesis, Produced, Variant Assay, Sequencing

    The c655 mutation disrupts TG transfer activity, but not PL transfer activity of the zebrafish Mtp complex. (A, B) COS-7 cells were first transfected with an expression vector for human APOB48 (5 μg), distributed equally in 6-well plates, and subsequently transfected with plasmids expressing either wild-type zebrafish mttp -FLAG, mttp stl -FLAG, mttp c655 -FLAG, or empty vector (pcDNA3) (3 μg). After 72 h, APOB48 was measured via ELISA in media (A) or in the cell (B). Data are representative of 7 independent experiments (each data point is the mean of three technical replicates), mean +/- SD, One-Way ANOVA with Bonferroni post-hoc tests, * p
    Figure Legend Snippet: The c655 mutation disrupts TG transfer activity, but not PL transfer activity of the zebrafish Mtp complex. (A, B) COS-7 cells were first transfected with an expression vector for human APOB48 (5 μg), distributed equally in 6-well plates, and subsequently transfected with plasmids expressing either wild-type zebrafish mttp -FLAG, mttp stl -FLAG, mttp c655 -FLAG, or empty vector (pcDNA3) (3 μg). After 72 h, APOB48 was measured via ELISA in media (A) or in the cell (B). Data are representative of 7 independent experiments (each data point is the mean of three technical replicates), mean +/- SD, One-Way ANOVA with Bonferroni post-hoc tests, * p

    Techniques Used: Mutagenesis, Activity Assay, Transfection, Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

    The opaque yolk phenotype results from the accumulation of aberrant cytoplasmic lipid droplets in the yolk syncytial layer. (A) (Top) Cartoon depicting the cross-sectional view of a 4 dpf zebrafish embryo. The YSL surrounds the yolk mass and serves as the embryonic digestive organ. The dashed box indicates the view expanded in the bottom panel and in panel B. (Bottom) Stored yolk lipids undergo lipolysis in yolk platelets (YP), presumably releasing free fatty acids into the YSL. These fatty acids are re-esterified in the ER bilayer to form TG, PL, and cholesterol esters. The lipids are packaged into B-lps in the ER with the help of Mtp and are likely further processed in the Golgi before being secreted into the perivitelline space (PS) and then circulation. (B) Representative transmission electron micrographs of the yolk and YSL from wild-type and mttp mutants; dashed lines delineate the YSL region, mt = mitochondria, scale = 10 μm. (C) Quantification of lipid droplet size in mttp mutants, n ≥ 700 lipid droplets in 2 fish per genotype; mean +/- SD. (D) Quantification of the number of lipid droplets per YSL area, n = 7–9 YSL regions per genotype (3–5 regions per fish, 2 fish per genotype); mean +/- SD, Kruskall-Wallis with Dunn’s Multiple Comparison test, vs. mttp c655/c655 , * p
    Figure Legend Snippet: The opaque yolk phenotype results from the accumulation of aberrant cytoplasmic lipid droplets in the yolk syncytial layer. (A) (Top) Cartoon depicting the cross-sectional view of a 4 dpf zebrafish embryo. The YSL surrounds the yolk mass and serves as the embryonic digestive organ. The dashed box indicates the view expanded in the bottom panel and in panel B. (Bottom) Stored yolk lipids undergo lipolysis in yolk platelets (YP), presumably releasing free fatty acids into the YSL. These fatty acids are re-esterified in the ER bilayer to form TG, PL, and cholesterol esters. The lipids are packaged into B-lps in the ER with the help of Mtp and are likely further processed in the Golgi before being secreted into the perivitelline space (PS) and then circulation. (B) Representative transmission electron micrographs of the yolk and YSL from wild-type and mttp mutants; dashed lines delineate the YSL region, mt = mitochondria, scale = 10 μm. (C) Quantification of lipid droplet size in mttp mutants, n ≥ 700 lipid droplets in 2 fish per genotype; mean +/- SD. (D) Quantification of the number of lipid droplets per YSL area, n = 7–9 YSL regions per genotype (3–5 regions per fish, 2 fish per genotype); mean +/- SD, Kruskall-Wallis with Dunn’s Multiple Comparison test, vs. mttp c655/c655 , * p

    Techniques Used: Transmission Assay, Fluorescence In Situ Hybridization

    The corresponding c655 mutation in human MTTP disrupts TG transfer but not PL transfer activity. (A) Immunofluorescence in COS-7 cells expressing wild-type human MTTP-FLAG or human MTTP(G865V)-FLAG proteins using anti-FLAG (red) and anti-Calnexin (green) antibodies; scale = 25 μm. (B) Human MTP-FLAG proteins (WT and G865V) were immunoprecipitated from COS-7 cell lysate (400 μg protein) using the M2 flag antibody and immunoblots were probed for both FLAG and PDI. For input, 15 μg protein was used. (C, D) COS-7 cells were co-transfected with human APOB48 and either wild-type human MTTP -FLAG, MTTP (G865V)-FLAG or empty pcDNA3 plasmids. After 72 h, APOB48 was measured via ELISA in media (C) or in the cell (D). Data are representative of 7 independent experiments (each data point is the mean of three technical replicates), pcDNA3 control data is re-graphed from Fig 5A 5B (data for Figs 5A , 5B , 6C and 6D were generated together); mean +/- SD, One-Way ANOVA with Bonferroni post-hoc tests, * p
    Figure Legend Snippet: The corresponding c655 mutation in human MTTP disrupts TG transfer but not PL transfer activity. (A) Immunofluorescence in COS-7 cells expressing wild-type human MTTP-FLAG or human MTTP(G865V)-FLAG proteins using anti-FLAG (red) and anti-Calnexin (green) antibodies; scale = 25 μm. (B) Human MTP-FLAG proteins (WT and G865V) were immunoprecipitated from COS-7 cell lysate (400 μg protein) using the M2 flag antibody and immunoblots were probed for both FLAG and PDI. For input, 15 μg protein was used. (C, D) COS-7 cells were co-transfected with human APOB48 and either wild-type human MTTP -FLAG, MTTP (G865V)-FLAG or empty pcDNA3 plasmids. After 72 h, APOB48 was measured via ELISA in media (C) or in the cell (D). Data are representative of 7 independent experiments (each data point is the mean of three technical replicates), pcDNA3 control data is re-graphed from Fig 5A 5B (data for Figs 5A , 5B , 6C and 6D were generated together); mean +/- SD, One-Way ANOVA with Bonferroni post-hoc tests, * p

    Techniques Used: Mutagenesis, Activity Assay, Immunofluorescence, Expressing, Immunoprecipitation, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Generated

    The c655 mutation supports secretion of small LDL-sized lipoproteins in vivo . (A) LipoGlo fish express the NanoLuc luciferase enzyme as a C-terminal fusion on ApoBb.1 as a result of TALEN-based genomic engineering [ 48 ]. (B) LipoGlo signal (RLU: relative luminescence units) in WT, mttp stl/stl , and mttp c655/c655 fish throughout embryonic development (2–6 dpf). Results represent pooled data from 3 independent experiments, n = 22–34 fish/genotype/time-point. Significance was determined with a Robust ANOVA, Games-Howell post-hoc tests were performed to compare genotypes at each day of development, and p-values were adjusted to control for multiple comparisons, a = WT vs. mttp stl/stl , p
    Figure Legend Snippet: The c655 mutation supports secretion of small LDL-sized lipoproteins in vivo . (A) LipoGlo fish express the NanoLuc luciferase enzyme as a C-terminal fusion on ApoBb.1 as a result of TALEN-based genomic engineering [ 48 ]. (B) LipoGlo signal (RLU: relative luminescence units) in WT, mttp stl/stl , and mttp c655/c655 fish throughout embryonic development (2–6 dpf). Results represent pooled data from 3 independent experiments, n = 22–34 fish/genotype/time-point. Significance was determined with a Robust ANOVA, Games-Howell post-hoc tests were performed to compare genotypes at each day of development, and p-values were adjusted to control for multiple comparisons, a = WT vs. mttp stl/stl , p

    Techniques Used: Mutagenesis, In Vivo, Fluorescence In Situ Hybridization, Luciferase

    10) Product Images from "Cell-Free Hepatitis B Virus Capsid Assembly Dependent on the Core Protein C-Terminal Domain and Regulated by Phosphorylation"

    Article Title: Cell-Free Hepatitis B Virus Capsid Assembly Dependent on the Core Protein C-Terminal Domain and Regulated by Phosphorylation

    Journal: Journal of Virology

    doi: 10.1128/JVI.00394-16

    HBV capsid assembly in RRL and effects of exogenous phosphatase and phosphatase inhibitors on assembly. The WT and mutant HBc proteins or the control luciferase (Luc) was translated in RRL. All samples were resolved by agarose gel electrophoresis. (A) The indicated protein translated in RRL was incubated overnight at 37°C in 1× NEB restriction digestion buffer 3 alone (lanes 1, 3, 5, 7, and 9) or with CIAP (lanes 2, 4, 6, 8, and 10) before resolution on the gel. The recombinant HBV capsid (rHBc) purified from E. coli was loaded in lane 11. (B) The indicated translation reaction mixture was loaded directly following translation upon dilution in double-distilled water (dH 2 O) and without the overnight incubation (i.e., no assembly reaction) (lanes 1, 5, 9, and 13), after dilution in NEB buffer 3 (buffer 3) but without the overnight incubation (lanes 2, 6, 10, and 14), after dilution in buffer 3 and with incubation overnight at 37°C (lanes 3, 7, 11, and 15), or after overnight incubation in buffer 3 and with CIAP (lanes 4, 8, 12, and 16). (C) The indicated translation reaction mixture was loaded directly following translation upon dilution in dH 2 O and without the overnight incubation (i.e., no assembly reaction) (lanes 1, 5, and 9), after dilution in dH 2 O and with incubation overnight at 37°C (lanes 2, 6, and 10), after dilution in buffer 3 and with incubation overnight at 37°C (lanes 3, 7, and 11), or after overnight incubation at 37°C in buffer 3 and with a mixture of phosphatase inhibitors (PPI) (lanes 4, 8, and 12). Each lane contained 3 μl translation product except that 3.125 ng rHBc was loaded in lane 11 of panel A. 35 S signals were detected by autoradiography (top). The HBc proteins were also detected by the MAb antibody against the NTD (bottom). C/3A/3E, WT, 3A, or 3E HBc subunits (i.e., not present in the capsid); C149, C-terminally truncated HBc protein (terminated at position 149); C-deP, dephosphorylated WT HBc subunits; Ca, capsids.
    Figure Legend Snippet: HBV capsid assembly in RRL and effects of exogenous phosphatase and phosphatase inhibitors on assembly. The WT and mutant HBc proteins or the control luciferase (Luc) was translated in RRL. All samples were resolved by agarose gel electrophoresis. (A) The indicated protein translated in RRL was incubated overnight at 37°C in 1× NEB restriction digestion buffer 3 alone (lanes 1, 3, 5, 7, and 9) or with CIAP (lanes 2, 4, 6, 8, and 10) before resolution on the gel. The recombinant HBV capsid (rHBc) purified from E. coli was loaded in lane 11. (B) The indicated translation reaction mixture was loaded directly following translation upon dilution in double-distilled water (dH 2 O) and without the overnight incubation (i.e., no assembly reaction) (lanes 1, 5, 9, and 13), after dilution in NEB buffer 3 (buffer 3) but without the overnight incubation (lanes 2, 6, 10, and 14), after dilution in buffer 3 and with incubation overnight at 37°C (lanes 3, 7, 11, and 15), or after overnight incubation in buffer 3 and with CIAP (lanes 4, 8, 12, and 16). (C) The indicated translation reaction mixture was loaded directly following translation upon dilution in dH 2 O and without the overnight incubation (i.e., no assembly reaction) (lanes 1, 5, and 9), after dilution in dH 2 O and with incubation overnight at 37°C (lanes 2, 6, and 10), after dilution in buffer 3 and with incubation overnight at 37°C (lanes 3, 7, and 11), or after overnight incubation at 37°C in buffer 3 and with a mixture of phosphatase inhibitors (PPI) (lanes 4, 8, and 12). Each lane contained 3 μl translation product except that 3.125 ng rHBc was loaded in lane 11 of panel A. 35 S signals were detected by autoradiography (top). The HBc proteins were also detected by the MAb antibody against the NTD (bottom). C/3A/3E, WT, 3A, or 3E HBc subunits (i.e., not present in the capsid); C149, C-terminally truncated HBc protein (terminated at position 149); C-deP, dephosphorylated WT HBc subunits; Ca, capsids.

    Techniques Used: Mutagenesis, Luciferase, Agarose Gel Electrophoresis, Incubation, Recombinant, Purification, Autoradiography

    Effects of exogenous phosphatase and RNase treatment on capsid assembly in RRL. The indicated HBc proteins were translated in RRL, and the translation reaction mixtures were resolved by agarose gel electrophoresis (top panels) or SDS-PAGE (bottom panels) without any further treatment (lanes 1, 7, 13, 19, 25, and 31) or were treated with NEB buffer 3 alone overnight at 37°C (buffer) (lanes 2, 8, 14, 20, 26, and 32), with buffer 3 plus CIAP overnight at 37°C (CIAP) (lanes 3, 9, 15, 21, 27, and 33), with buffer 3 plus CIAP overnight at 37°C followed by RNase treatment for one additional hour (CIAP-RNase) (lanes 4, 10, 16, 22, 28, and 34), with RNase for 1 h followed by buffer 3 plus CIAP overnight at 37°C (lanes 5, 11, 17, 23, 29, and 35), or with the mixture of phosphatase inhibitors overnight at 37°C (lanes 6, 12, 18, 24, 30, and 36). All lanes contained 2 μl translation products. 35 S-labeled HBc proteins were detected by autoradiography. C, 3A, and 3E/7A, WT or mutant HBc subunits; C149, C-terminally truncated HBc protein (terminated at position 149); C-deP, dephosphorylated HBc subunits; Ca, capsids.
    Figure Legend Snippet: Effects of exogenous phosphatase and RNase treatment on capsid assembly in RRL. The indicated HBc proteins were translated in RRL, and the translation reaction mixtures were resolved by agarose gel electrophoresis (top panels) or SDS-PAGE (bottom panels) without any further treatment (lanes 1, 7, 13, 19, 25, and 31) or were treated with NEB buffer 3 alone overnight at 37°C (buffer) (lanes 2, 8, 14, 20, 26, and 32), with buffer 3 plus CIAP overnight at 37°C (CIAP) (lanes 3, 9, 15, 21, 27, and 33), with buffer 3 plus CIAP overnight at 37°C followed by RNase treatment for one additional hour (CIAP-RNase) (lanes 4, 10, 16, 22, 28, and 34), with RNase for 1 h followed by buffer 3 plus CIAP overnight at 37°C (lanes 5, 11, 17, 23, 29, and 35), or with the mixture of phosphatase inhibitors overnight at 37°C (lanes 6, 12, 18, 24, 30, and 36). All lanes contained 2 μl translation products. 35 S-labeled HBc proteins were detected by autoradiography. C, 3A, and 3E/7A, WT or mutant HBc subunits; C149, C-terminally truncated HBc protein (terminated at position 149); C-deP, dephosphorylated HBc subunits; Ca, capsids.

    Techniques Used: Agarose Gel Electrophoresis, SDS Page, Labeling, Autoradiography, Mutagenesis

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    Article Snippet: .. The λ-DNA Hind digest (New England Biolabs) was stained with the intercalating fluorescence dye YOYO-1 (Invitrogen) at a dye-to-base pair ratio of ~1:10 in 5× TBE buffer. ..

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    Purification:

    Article Title: Efficient modification of λ-DNA substrates for single-molecule studies
    Article Snippet: .. Inserting synthetic oligonucleotides into λ-DNA Recombinant λ-DNA was obtained from strain IF189, which was modified and purified as described above, and 25 μg of the DNA was incubated with 150 U of Nt.BspQI (NEB# R0644S) in a 250 µL reaction with 1X buffer 3.1 (NEB #B7203) at 55 °C for 1 hour. .. The reaction was halted with 1 U of proteinase K (NEB #P8107S) for 1 hour at 55 °C.

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    Article Title: Efficient modification of λ-DNA substrates for single-molecule studies
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    Staining:

    Article Title: Massively-Parallel Ultra-High-Aspect-Ratio Nanochannels as Mesoporous Membranes
    Article Snippet: .. The λ-DNA Hind digest (New England Biolabs) was stained with the intercalating fluorescence dye YOYO-1 (Invitrogen) at a dye-to-base pair ratio of ~1:10 in 5× TBE buffer. ..

    Recombinant:

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    Article Title: Signal and noise in bridging PCR
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    New England Biolabs nebuffer 3 1
    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).
    Nebuffer 3 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 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

    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: 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

    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.

    Journal: Sensors and Actuators. B, Chemical

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

    doi: 10.1016/j.snb.2018.06.069

    Figure Lengend 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.

    Article Snippet: 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.

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