β catenin lentivirus vector  (New England Biolabs)


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    Name:
    BamHI
    Description:
    BamHI 50 000 units
    Catalog Number:
    r0136l
    Price:
    249
    Size:
    50 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs β catenin lentivirus vector
    BamHI
    BamHI 50 000 units
    https://www.bioz.com/result/β catenin lentivirus vector/product/New England Biolabs
    Average 90 stars, based on 214 article reviews
    Price from $9.99 to $1999.99
    β catenin lentivirus vector - by Bioz Stars, 2021-02
    90/100 stars

    Images

    1) Product Images from "β-catenin promotes intracellular bacterial killing via suppression of Pseudomonas aeruginosa-triggered macrophage autophagy"

    Article Title: β-catenin promotes intracellular bacterial killing via suppression of Pseudomonas aeruginosa-triggered macrophage autophagy

    Journal: The Journal of International Medical Research

    doi: 10.1177/0300060517692147

    RAW264.7 cells transduced with β-catenin (encoded by catenin beta 1 [ CTNNB1 ] gene) or controls were infected with Pseudomonas aeruginosa at a multiplicity of infection value of 1 and showed that β-catenin suppressed P. aeruginosa -triggered macrophage autophagy: (a) Representative Western blots showing microtubule-associated protein 1 light chain 3 alpha (LC3)-I, LC3-II and β-catenin protein levels with or without P. aeruginosa infection for 1, 2, 4 or 8 h (LC3-II:β-actin ratio shown underneath); (b) LC3 relative integrated density values normalized to β-actin, presented as ratio of control transduced cells showing lower LC3 levels in β-catenin versus control cells (* P
    Figure Legend Snippet: RAW264.7 cells transduced with β-catenin (encoded by catenin beta 1 [ CTNNB1 ] gene) or controls were infected with Pseudomonas aeruginosa at a multiplicity of infection value of 1 and showed that β-catenin suppressed P. aeruginosa -triggered macrophage autophagy: (a) Representative Western blots showing microtubule-associated protein 1 light chain 3 alpha (LC3)-I, LC3-II and β-catenin protein levels with or without P. aeruginosa infection for 1, 2, 4 or 8 h (LC3-II:β-actin ratio shown underneath); (b) LC3 relative integrated density values normalized to β-actin, presented as ratio of control transduced cells showing lower LC3 levels in β-catenin versus control cells (* P

    Techniques Used: Transduction, Infection, Western Blot

    Representative Western blots of β-catenin levels from murine macrophage-like RAW264.7 cells infected with Pseudomonas aeruginosa at multiplicity of infection (MOI) values of 0.1–50 for (a) 6 h or (b) 12 h; Whole cell β-catenin relative integrated density values normalized to β-actin, presented as percentage of control values for (c) cells infected for 6 h and (d) cells infected for 12 h (β-catenin levels reduced with increasing P. aeruginosa multiplicity of infection values); Cell viability of RAW264.7 cells infected with P. aeruginosa at MOI values of 0.1–50 for (e) 6 h or (f) 12 h, evaluated by cell counting kit-8 assay. Data presented as mean ± SD of three individual experiments; *** P
    Figure Legend Snippet: Representative Western blots of β-catenin levels from murine macrophage-like RAW264.7 cells infected with Pseudomonas aeruginosa at multiplicity of infection (MOI) values of 0.1–50 for (a) 6 h or (b) 12 h; Whole cell β-catenin relative integrated density values normalized to β-actin, presented as percentage of control values for (c) cells infected for 6 h and (d) cells infected for 12 h (β-catenin levels reduced with increasing P. aeruginosa multiplicity of infection values); Cell viability of RAW264.7 cells infected with P. aeruginosa at MOI values of 0.1–50 for (e) 6 h or (f) 12 h, evaluated by cell counting kit-8 assay. Data presented as mean ± SD of three individual experiments; *** P

    Techniques Used: Western Blot, Infection, Cell Counting

    Construction of murine macrophage-like RAW264.7 cells with stable catenin beta 1 expression: (a) Flow cytometry scatter plot showing RAW264.7 cells transduced with control lentivirus or β-catenin lentivirus, sorted according to mCherry marker expression; (b) Representative Western blot showing β-catenin levels in control and β-catenin-transduced monoclones; (c) β-catenin relative integrated density values normalized to β-actin, presented as percentage of control transduced cells (*** P
    Figure Legend Snippet: Construction of murine macrophage-like RAW264.7 cells with stable catenin beta 1 expression: (a) Flow cytometry scatter plot showing RAW264.7 cells transduced with control lentivirus or β-catenin lentivirus, sorted according to mCherry marker expression; (b) Representative Western blot showing β-catenin levels in control and β-catenin-transduced monoclones; (c) β-catenin relative integrated density values normalized to β-actin, presented as percentage of control transduced cells (*** P

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Transduction, Marker, Western Blot

    RAW264.7 cells transduced with β-catenin (encoded by catenin beta 1 [ CTNNB1 ] gene) or controls were infected with Pseudomonas aeruginosa : (a) Plate counts showed that bacterial killing was increased in cells overexpressing CTNNB1 versus controls at 1 h (*** P
    Figure Legend Snippet: RAW264.7 cells transduced with β-catenin (encoded by catenin beta 1 [ CTNNB1 ] gene) or controls were infected with Pseudomonas aeruginosa : (a) Plate counts showed that bacterial killing was increased in cells overexpressing CTNNB1 versus controls at 1 h (*** P

    Techniques Used: Transduction, Infection

    Representative Western blots of β-catenin levels from murine macrophage-like RAW264.7 cells infected with Pseudomonas aeruginosa at multiplicity of infection (MOI) values of (a) 1 and (b) 5, for 6–48 h; (c) Representative Western blots of nuclear and cytosolic β-catenin levels following RAW264.7 cell infection with P. aeruginosa at a MOI of 1 for 6–48 h, showing increased nuclear β-catenin levels at 6, 12 and 24 h and decreased levels at 48 h versus controls, and decreased cytosolic β-catenin levels at all time-points versus controls; Whole cell β-catenin relative integrated density values normalized to β-actin, presented as percentage of control values for (d) P. aeruginosa MOI 1 and (e) P. aeruginosa MOI 5 showing β-catenin levels increased at early time-points then reduced in a time-dependent manner following P. aeruginosa infection. Data presented as mean ± SD of three individual experiments; *** P
    Figure Legend Snippet: Representative Western blots of β-catenin levels from murine macrophage-like RAW264.7 cells infected with Pseudomonas aeruginosa at multiplicity of infection (MOI) values of (a) 1 and (b) 5, for 6–48 h; (c) Representative Western blots of nuclear and cytosolic β-catenin levels following RAW264.7 cell infection with P. aeruginosa at a MOI of 1 for 6–48 h, showing increased nuclear β-catenin levels at 6, 12 and 24 h and decreased levels at 48 h versus controls, and decreased cytosolic β-catenin levels at all time-points versus controls; Whole cell β-catenin relative integrated density values normalized to β-actin, presented as percentage of control values for (d) P. aeruginosa MOI 1 and (e) P. aeruginosa MOI 5 showing β-catenin levels increased at early time-points then reduced in a time-dependent manner following P. aeruginosa infection. Data presented as mean ± SD of three individual experiments; *** P

    Techniques Used: Western Blot, Infection

    RAW264.7 cells transduced with β-catenin (encoded by catenin beta 1 [ CTNNB1 ] gene) or control cells were infected with Pseudomonas aeruginosa at a multiplicity of infection value of 10 for 1 and 2 h: Microtubule-associated protein 1 light chain 3 alpha (LC3) protein levels were analysed by Western blot to confirm autophagy induction by (a and c) rapamycin and (b and d) starvation, with LC3-II:β-actin ratios shown (both *** P
    Figure Legend Snippet: RAW264.7 cells transduced with β-catenin (encoded by catenin beta 1 [ CTNNB1 ] gene) or control cells were infected with Pseudomonas aeruginosa at a multiplicity of infection value of 10 for 1 and 2 h: Microtubule-associated protein 1 light chain 3 alpha (LC3) protein levels were analysed by Western blot to confirm autophagy induction by (a and c) rapamycin and (b and d) starvation, with LC3-II:β-actin ratios shown (both *** P

    Techniques Used: Transduction, Infection, Western Blot

    2) Product Images from "Novel N4-Like Bacteriophages of Pectobacterium atrosepticum"

    Article Title: Novel N4-Like Bacteriophages of Pectobacterium atrosepticum

    Journal: Pharmaceuticals

    doi: 10.3390/ph11020045

    Genomic DNA of Pectobacterium phages CB1, CB3, and CB4, BamHI-digested (lanes 3, 5, and 7, respectively) and undigested (lanes 2, 4, and 6, respectively). Lane 1, DNA marker (Hyperladder 1 kb, Bioline). Gel concentration 1% w / v agarose.
    Figure Legend Snippet: Genomic DNA of Pectobacterium phages CB1, CB3, and CB4, BamHI-digested (lanes 3, 5, and 7, respectively) and undigested (lanes 2, 4, and 6, respectively). Lane 1, DNA marker (Hyperladder 1 kb, Bioline). Gel concentration 1% w / v agarose.

    Techniques Used: Marker, Concentration Assay

    3) Product Images from "Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins"

    Article Title: Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202138

    Sites of structural changes induced by the hyper-negative supercoiling detected by Nuclease SI. (A) Experimental scheme. The red-filled circle designates 32 P. The different steps of the experiment are indicated: first (1), the digestion by the Nuclease SI; second (2), the digestion by (BamHI + BglII) or (BahmHI + HindIII); third (3), electrophoresis on a sequencing gel. (B) The enzymatic probe used to map the fine structure of the T -2 and T -6 topoisomers is Nuclease SI. Nuclease SI is at 2 mU microL -1 and DNA at 0.5 nM. After the Nuclease SI reaction, the samples are treated to remove the proteins. The DNAs are precipitated and submitted to the BamHI+HindIII double digestion to only visualize DNA fragments from one of the two radiolabeled strands. The reaction products are analyzed on two different sequencing gels (8% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 5) or the guanines and adenines (G+A lanes; lanes 2 and 6). (C) Same as 3B except that the samples are submitted to the BglII+BamHI double digestion to only visualize DNA fragments from the complementary radiolabeled strands. The reaction products are analyzed on two different sequencing gels (7% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 7) or the guanines and adenines (G+A lanes; lanes 2 and 6).
    Figure Legend Snippet: Sites of structural changes induced by the hyper-negative supercoiling detected by Nuclease SI. (A) Experimental scheme. The red-filled circle designates 32 P. The different steps of the experiment are indicated: first (1), the digestion by the Nuclease SI; second (2), the digestion by (BamHI + BglII) or (BahmHI + HindIII); third (3), electrophoresis on a sequencing gel. (B) The enzymatic probe used to map the fine structure of the T -2 and T -6 topoisomers is Nuclease SI. Nuclease SI is at 2 mU microL -1 and DNA at 0.5 nM. After the Nuclease SI reaction, the samples are treated to remove the proteins. The DNAs are precipitated and submitted to the BamHI+HindIII double digestion to only visualize DNA fragments from one of the two radiolabeled strands. The reaction products are analyzed on two different sequencing gels (8% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 5) or the guanines and adenines (G+A lanes; lanes 2 and 6). (C) Same as 3B except that the samples are submitted to the BglII+BamHI double digestion to only visualize DNA fragments from the complementary radiolabeled strands. The reaction products are analyzed on two different sequencing gels (7% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 7) or the guanines and adenines (G+A lanes; lanes 2 and 6).

    Techniques Used: Electrophoresis, Sequencing

    4) Product Images from "The P-SSP7 Cyanophage Has a Linear Genome with Direct Terminal Repeats"

    Article Title: The P-SSP7 Cyanophage Has a Linear Genome with Direct Terminal Repeats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036710

    Digestion and Southern analyses of the P-SSP7 genome. (A) Schematic genome map showing the positions of the restriction enzyme cleavage sites (red) and the expected fragment sizes after digestion with BamHI alone (top) and both BamHI and PmeI (bottom) based on the revised genome arrangement shown in Fig. 1C. (B) Restriction digestion of the P-SSP7 genome extracted from phage particles (lanes 3 and 4) and the genome cloned into a fosmid (lanes 5 and 6), with BamHI alone (lanes 3 and 5) or with BamHI and PmeI (lanes 4 and 6), separated by pulse field gel electrophoresis. Note that the only difference for digestion of the cloned genome is the presence of an additional fragment corresponding to the size of the fosmid vector. Fragments corresponding to the expected sizes shown in (A) are marked with the appropriate letter designations (a to f). Fragment size markers (M): 1 kb DNA ladder (lane 1) and Lambda DNA cut with HindIII (lane 2), are shown. (C) Southern analyses of the restriction digested DNA in (B) using 4 probes (denoted above the lanes) show that the repeat region appears twice on the genome on the same fragments as the first and last ORFs. The positions of the gene probes on the genome are shown as light blue boxes and the repeat region probe as green boxes in the top panel of (A). Lane numbering and fragment designations are the same as in (B).
    Figure Legend Snippet: Digestion and Southern analyses of the P-SSP7 genome. (A) Schematic genome map showing the positions of the restriction enzyme cleavage sites (red) and the expected fragment sizes after digestion with BamHI alone (top) and both BamHI and PmeI (bottom) based on the revised genome arrangement shown in Fig. 1C. (B) Restriction digestion of the P-SSP7 genome extracted from phage particles (lanes 3 and 4) and the genome cloned into a fosmid (lanes 5 and 6), with BamHI alone (lanes 3 and 5) or with BamHI and PmeI (lanes 4 and 6), separated by pulse field gel electrophoresis. Note that the only difference for digestion of the cloned genome is the presence of an additional fragment corresponding to the size of the fosmid vector. Fragments corresponding to the expected sizes shown in (A) are marked with the appropriate letter designations (a to f). Fragment size markers (M): 1 kb DNA ladder (lane 1) and Lambda DNA cut with HindIII (lane 2), are shown. (C) Southern analyses of the restriction digested DNA in (B) using 4 probes (denoted above the lanes) show that the repeat region appears twice on the genome on the same fragments as the first and last ORFs. The positions of the gene probes on the genome are shown as light blue boxes and the repeat region probe as green boxes in the top panel of (A). Lane numbering and fragment designations are the same as in (B).

    Techniques Used: Clone Assay, Nucleic Acid Electrophoresis, Plasmid Preparation, Lambda DNA Preparation

    Schematic illustration of the arrangement of the P-SSP7 genome. (A) Sequencing of the ends of the P-SSP7 genome extracted directly from phage particles. Arrows, and numbers under the arrows, indicate the sequences acquired: Blue from the entire genome and green from end fragments produced by digestion of the genome with the BamHI and PmeI restriction enzymes. The positions of the primers used for sequencing are shown in black type at the beginning of the arrows. Genome numbering for the primers and sequences is that for the originally published sequence [5] . The purple line denotes the 728 bp region found to be upstream of ORF1 in this study, but positioned downstream of ORF54 in the originally published sequence. The repeat regions are shown in red at both ends of the genome. (B) Diagram showing the arrangement of the P-SSP7 genome as originally published (GenBank accession numbers: AY939843.1, [5] and GU071093 [16] . (C) Diagram of the revised genome arrangement based on the results from this study (updated GeneBank submission, accession number: AY939843.2).
    Figure Legend Snippet: Schematic illustration of the arrangement of the P-SSP7 genome. (A) Sequencing of the ends of the P-SSP7 genome extracted directly from phage particles. Arrows, and numbers under the arrows, indicate the sequences acquired: Blue from the entire genome and green from end fragments produced by digestion of the genome with the BamHI and PmeI restriction enzymes. The positions of the primers used for sequencing are shown in black type at the beginning of the arrows. Genome numbering for the primers and sequences is that for the originally published sequence [5] . The purple line denotes the 728 bp region found to be upstream of ORF1 in this study, but positioned downstream of ORF54 in the originally published sequence. The repeat regions are shown in red at both ends of the genome. (B) Diagram showing the arrangement of the P-SSP7 genome as originally published (GenBank accession numbers: AY939843.1, [5] and GU071093 [16] . (C) Diagram of the revised genome arrangement based on the results from this study (updated GeneBank submission, accession number: AY939843.2).

    Techniques Used: Sequencing, Produced

    Digestion and Southern analyses of the P-SSP7 genome. (A) Schematic genome map showing the positions of the restriction enzyme cleavage sites (red) and the expected fragment sizes after digestion with BamHI alone (top) and both BamHI and PmeI (bottom) based on the revised genome arrangement shown in Fig. 1C. (B) Restriction digestion of the P-SSP7 genome extracted from phage particles (lanes 3 and 4) and the genome cloned into a fosmid (lanes 5 and 6), with BamHI alone (lanes 3 and 5) or with BamHI and PmeI (lanes 4 and 6), separated by pulse field gel electrophoresis. Note that the only difference for digestion of the cloned genome is the presence of an additional fragment corresponding to the size of the fosmid vector. Fragments corresponding to the expected sizes shown in (A) are marked with the appropriate letter designations (a to f). Fragment size markers (M): 1 kb DNA ladder (lane 1) and Lambda DNA cut with HindIII (lane 2), are shown. (C) Southern analyses of the restriction digested DNA in (B) using 4 probes (denoted above the lanes) show that the repeat region appears twice on the genome on the same fragments as the first and last ORFs. The positions of the gene probes on the genome are shown as light blue boxes and the repeat region probe as green boxes in the top panel of (A). Lane numbering and fragment designations are the same as in (B).
    Figure Legend Snippet: Digestion and Southern analyses of the P-SSP7 genome. (A) Schematic genome map showing the positions of the restriction enzyme cleavage sites (red) and the expected fragment sizes after digestion with BamHI alone (top) and both BamHI and PmeI (bottom) based on the revised genome arrangement shown in Fig. 1C. (B) Restriction digestion of the P-SSP7 genome extracted from phage particles (lanes 3 and 4) and the genome cloned into a fosmid (lanes 5 and 6), with BamHI alone (lanes 3 and 5) or with BamHI and PmeI (lanes 4 and 6), separated by pulse field gel electrophoresis. Note that the only difference for digestion of the cloned genome is the presence of an additional fragment corresponding to the size of the fosmid vector. Fragments corresponding to the expected sizes shown in (A) are marked with the appropriate letter designations (a to f). Fragment size markers (M): 1 kb DNA ladder (lane 1) and Lambda DNA cut with HindIII (lane 2), are shown. (C) Southern analyses of the restriction digested DNA in (B) using 4 probes (denoted above the lanes) show that the repeat region appears twice on the genome on the same fragments as the first and last ORFs. The positions of the gene probes on the genome are shown as light blue boxes and the repeat region probe as green boxes in the top panel of (A). Lane numbering and fragment designations are the same as in (B).

    Techniques Used: Clone Assay, Nucleic Acid Electrophoresis, Plasmid Preparation, Lambda DNA Preparation

    5) Product Images from "Chlamydomonas reinhardtii hydin is a central pair protein required for flagellar motility"

    Article Title: Chlamydomonas reinhardtii hydin is a central pair protein required for flagellar motility

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200611115

    HY3 gene-silencing vector and anti-hydin antibody. (A) C. reinhardtii HY3 , which encodes hydin, is a gene of 17.7 kb. Fragment A, corresponding to exon 3 of HY3 , and a BamHI–SalI piece of fragment A were cloned into bacterial expression vectors, and the fusion proteins were used for antibody production and purification. A gene-silencing vector was constructed from fragment A, fragment S (another PCR product of HY3 ), a triple HA tag, and the promoter and terminator region of the LC8 gene. (B) Coomassie-stained gel (a; 4–20% SDS-PAGE) and Western blot (b; 7.5% SDS-PAGE) of isolated axonemes of CC3395 (control) and the HY3 RNAi strains hyN3 and hyN4. Anti-hydin specifically stained a band of ∼540 kD that was strongly reduced in the HY3 RNAi strains. (C) Western blots probed with anti-hydin and anti-IFT172 ( Cole et al., 1998 ) comparing the amount of hydin present in deflagellated cells (CB) and isolated flagella (Fla) or axonemes (Ax). (a) Equivalent numbers of cell bodies and flagellar pairs from ∼10 6 cells were loaded. (b) Equal amounts (∼25 μg) of cell body and axonemal protein were loaded. IFT172, an intraflagellar transport protein used as a control, is present in the cell body and flagella; a considerable amount remains with the axonemes ( Hou et al., 2004 ). (D) Immunofluorescence images of methanol-fixed cells of strains CC3395 (control), hyN4, and hyS2 labeled with anti-acetylated tubulin (a, d, and g) and anti-hydin (b, e, and h). Merged images (c, f, and i) reveal the localization of hydin to the flagella of wild-type cells and the reduction of hydin in the hydin RNAi cells. Note the shorter flagella in the latter. At least part of the fluorescence in the cell bodies stained with anti-hydin is background caused by chlorophyll autofluorescence. Bar, 5 μm.
    Figure Legend Snippet: HY3 gene-silencing vector and anti-hydin antibody. (A) C. reinhardtii HY3 , which encodes hydin, is a gene of 17.7 kb. Fragment A, corresponding to exon 3 of HY3 , and a BamHI–SalI piece of fragment A were cloned into bacterial expression vectors, and the fusion proteins were used for antibody production and purification. A gene-silencing vector was constructed from fragment A, fragment S (another PCR product of HY3 ), a triple HA tag, and the promoter and terminator region of the LC8 gene. (B) Coomassie-stained gel (a; 4–20% SDS-PAGE) and Western blot (b; 7.5% SDS-PAGE) of isolated axonemes of CC3395 (control) and the HY3 RNAi strains hyN3 and hyN4. Anti-hydin specifically stained a band of ∼540 kD that was strongly reduced in the HY3 RNAi strains. (C) Western blots probed with anti-hydin and anti-IFT172 ( Cole et al., 1998 ) comparing the amount of hydin present in deflagellated cells (CB) and isolated flagella (Fla) or axonemes (Ax). (a) Equivalent numbers of cell bodies and flagellar pairs from ∼10 6 cells were loaded. (b) Equal amounts (∼25 μg) of cell body and axonemal protein were loaded. IFT172, an intraflagellar transport protein used as a control, is present in the cell body and flagella; a considerable amount remains with the axonemes ( Hou et al., 2004 ). (D) Immunofluorescence images of methanol-fixed cells of strains CC3395 (control), hyN4, and hyS2 labeled with anti-acetylated tubulin (a, d, and g) and anti-hydin (b, e, and h). Merged images (c, f, and i) reveal the localization of hydin to the flagella of wild-type cells and the reduction of hydin in the hydin RNAi cells. Note the shorter flagella in the latter. At least part of the fluorescence in the cell bodies stained with anti-hydin is background caused by chlorophyll autofluorescence. Bar, 5 μm.

    Techniques Used: Plasmid Preparation, Clone Assay, Expressing, Purification, Construct, Polymerase Chain Reaction, Staining, SDS Page, Western Blot, Isolation, Immunofluorescence, Labeling, Fluorescence

    6) Product Images from "Genome-Scale Identification of Resistance Functions in Pseudomonas aeruginosa Using Tn-seq"

    Article Title: Genome-Scale Identification of Resistance Functions in Pseudomonas aeruginosa Using Tn-seq

    Journal: mBio

    doi: 10.1128/mBio.00315-10

    Tn-seq circle method. The steps used to amplify and sequence transposon insertion junctions are illustrated, beginning with a DNA fragment carrying a transposon insertion (top). First, total DNA from a mutant pool is sheared and end repaired, and one Illumina adaptor (A2) is ligated to all free ends (step 1). The sample is then digested with a restriction enzyme that cuts near one transposon end (in this work, BamHI, which cuts 114 bp from the transposon’s left end) (step 2). Following a size selection step, single-strand fragments which include the transposon end are circularized by templated ligation (step 3). Oligo, oligonucleotide. Fragments which have not circularized (representing most of the DNA in the sample) are degraded in a subsequent exonuclease step (step 4). The transposon-genome junctions from the circularized fragments are then amplified by quantitative PCR in a step in which the second required Illumina adaptor (A1) is introduced (step 5). The products are sequenced on an Illumina flow cell using a sequencing primer corresponding to the transposon end (Seq), and each sequence read is then mapped to the genome (step 6).
    Figure Legend Snippet: Tn-seq circle method. The steps used to amplify and sequence transposon insertion junctions are illustrated, beginning with a DNA fragment carrying a transposon insertion (top). First, total DNA from a mutant pool is sheared and end repaired, and one Illumina adaptor (A2) is ligated to all free ends (step 1). The sample is then digested with a restriction enzyme that cuts near one transposon end (in this work, BamHI, which cuts 114 bp from the transposon’s left end) (step 2). Following a size selection step, single-strand fragments which include the transposon end are circularized by templated ligation (step 3). Oligo, oligonucleotide. Fragments which have not circularized (representing most of the DNA in the sample) are degraded in a subsequent exonuclease step (step 4). The transposon-genome junctions from the circularized fragments are then amplified by quantitative PCR in a step in which the second required Illumina adaptor (A1) is introduced (step 5). The products are sequenced on an Illumina flow cell using a sequencing primer corresponding to the transposon end (Seq), and each sequence read is then mapped to the genome (step 6).

    Techniques Used: Sequencing, Mutagenesis, Selection, Ligation, Amplification, Real-time Polymerase Chain Reaction

    7) Product Images from "Recognition of DNA Termini by the C-Terminal Region of the Ku80 and the DNA-Dependent Protein Kinase Catalytic Subunit"

    Article Title: Recognition of DNA Termini by the C-Terminal Region of the Ku80 and the DNA-Dependent Protein Kinase Catalytic Subunit

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0127321

    Distinct Influences of the Ku80 C-terminus on DNA-PK Activation with Linearized Plasmid DNA. a) DNA-PK kinase stimulation with plasmid DNA linearized with EcoRV generating blunt-ended termini and with KpnI generating 4 base 3’ single stranded overhangs. b) DNA-PK kinase stimulation with plasmid DNA linearized with XhoI and BamHI generating 4 base 5’ single stranded overhangs. DNA substrates are depicted pictorially. DNA termini generated by digestion are depicted below each graph indicating locations of pyrimidines (Py) and purines (Pu). Kinase activity is reported as the mean and SD of pmol of phosphate transferred. Asterisks indicate statistically significant differences compared to wild type (p
    Figure Legend Snippet: Distinct Influences of the Ku80 C-terminus on DNA-PK Activation with Linearized Plasmid DNA. a) DNA-PK kinase stimulation with plasmid DNA linearized with EcoRV generating blunt-ended termini and with KpnI generating 4 base 3’ single stranded overhangs. b) DNA-PK kinase stimulation with plasmid DNA linearized with XhoI and BamHI generating 4 base 5’ single stranded overhangs. DNA substrates are depicted pictorially. DNA termini generated by digestion are depicted below each graph indicating locations of pyrimidines (Py) and purines (Pu). Kinase activity is reported as the mean and SD of pmol of phosphate transferred. Asterisks indicate statistically significant differences compared to wild type (p

    Techniques Used: Activation Assay, Plasmid Preparation, Generated, Activity Assay

    8) Product Images from "The development and application of new crystallization method for tobacco mosaic virus coat protein"

    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein

    Journal: Virology Journal

    doi: 10.1186/1743-422X-9-279

    TMV-CP DNA fragments (1% agarose gel). ( A ) The whole TMV-CP fragments with BamH I and Xho I restriction enzyme cutting sites which have been cloned in PGEX-6P-1. As shown in lane 1, amplified PCR product ran at approximately 500 bp compared with the DNA marker (lane M). Lane 2 is a positive control with DNA template. Lane 3 is a negative control without DNA template. ( B ) The whole TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. As shown in lane 1, amplified PCR product ran at approximately 500 bp compared with the DNA marker (lane M). Lane 2 is a negative control without DNA template. Lane 3 is a positive control with DNA template. ( C ) The truncation of four amino acids from the C-terminus of TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. Lane 1 is a negative control without DNA template, whereas lane 2 is a positive control with DNA template. Lane 3 is the amplified PCR product that ran at approximately 500 bp compared with the DNA marker (lane M).
    Figure Legend Snippet: TMV-CP DNA fragments (1% agarose gel). ( A ) The whole TMV-CP fragments with BamH I and Xho I restriction enzyme cutting sites which have been cloned in PGEX-6P-1. As shown in lane 1, amplified PCR product ran at approximately 500 bp compared with the DNA marker (lane M). Lane 2 is a positive control with DNA template. Lane 3 is a negative control without DNA template. ( B ) The whole TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. As shown in lane 1, amplified PCR product ran at approximately 500 bp compared with the DNA marker (lane M). Lane 2 is a negative control without DNA template. Lane 3 is a positive control with DNA template. ( C ) The truncation of four amino acids from the C-terminus of TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. Lane 1 is a negative control without DNA template, whereas lane 2 is a positive control with DNA template. Lane 3 is the amplified PCR product that ran at approximately 500 bp compared with the DNA marker (lane M).

    Techniques Used: Agarose Gel Electrophoresis, Clone Assay, Amplification, Polymerase Chain Reaction, Marker, Positive Control, Negative Control

    9) Product Images from "Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae"

    Article Title: Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae

    Journal: Bioengineered

    doi: 10.4161/bioe.29167

    Figure 4. In vivo assembly of synthetic genes by IVOE. Each overlapping region allowed crossover events to occur between fragments giving rise to an autonomously repaired vector containing single and double expression cassettes in the correct orientation. ( A ) and ( B ) Single expression cassettes are constructed in pESC by engineering specific primers containing overhangs to foster in vivo cloning with the linearized plasmid in yeast (pJRoC30 was used as template for Vp or Lac amplifications). ( C ) and ( D ) The pESC constructs obtained in ( A ) and ( B ) were used as scaffolds to assemble Lac and Vp genes under the control of different promoter/terminator pairs. Primers used: (1)-MCS1- Vp / Lac -α-BamHI, (2)-MCS2- Vp -ter-NheI, (3)-MCS2- Lac -ter-NheI, (4)-MCS1- Vp / Lac -α-SpeI, (5)-MCS1- Lac -ter-SacI and (6)-MCS1- Vp -ter-SacI. Black arrows indicate the direction of the transcription process.
    Figure Legend Snippet: Figure 4. In vivo assembly of synthetic genes by IVOE. Each overlapping region allowed crossover events to occur between fragments giving rise to an autonomously repaired vector containing single and double expression cassettes in the correct orientation. ( A ) and ( B ) Single expression cassettes are constructed in pESC by engineering specific primers containing overhangs to foster in vivo cloning with the linearized plasmid in yeast (pJRoC30 was used as template for Vp or Lac amplifications). ( C ) and ( D ) The pESC constructs obtained in ( A ) and ( B ) were used as scaffolds to assemble Lac and Vp genes under the control of different promoter/terminator pairs. Primers used: (1)-MCS1- Vp / Lac -α-BamHI, (2)-MCS2- Vp -ter-NheI, (3)-MCS2- Lac -ter-NheI, (4)-MCS1- Vp / Lac -α-SpeI, (5)-MCS1- Lac -ter-SacI and (6)-MCS1- Vp -ter-SacI. Black arrows indicate the direction of the transcription process.

    Techniques Used: In Vivo, Plasmid Preparation, Expressing, Construct, Clone Assay

    10) Product Images from "Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins"

    Article Title: Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202138

    Sites of structural changes induced by the hyper-negative supercoiling detected by Nuclease SI. (A) Experimental scheme. The red-filled circle designates 32 P. The different steps of the experiment are indicated: first (1), the digestion by the Nuclease SI; second (2), the digestion by (BamHI + BglII) or (BahmHI + HindIII); third (3), electrophoresis on a sequencing gel. (B) The enzymatic probe used to map the fine structure of the T -2 and T -6 topoisomers is Nuclease SI. Nuclease SI is at 2 mU microL -1 and DNA at 0.5 nM. After the Nuclease SI reaction, the samples are treated to remove the proteins. The DNAs are precipitated and submitted to the BamHI+HindIII double digestion to only visualize DNA fragments from one of the two radiolabeled strands. The reaction products are analyzed on two different sequencing gels (8% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 5) or the guanines and adenines (G+A lanes; lanes 2 and 6). (C) Same as 3B except that the samples are submitted to the BglII+BamHI double digestion to only visualize DNA fragments from the complementary radiolabeled strands. The reaction products are analyzed on two different sequencing gels (7% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 7) or the guanines and adenines (G+A lanes; lanes 2 and 6).
    Figure Legend Snippet: Sites of structural changes induced by the hyper-negative supercoiling detected by Nuclease SI. (A) Experimental scheme. The red-filled circle designates 32 P. The different steps of the experiment are indicated: first (1), the digestion by the Nuclease SI; second (2), the digestion by (BamHI + BglII) or (BahmHI + HindIII); third (3), electrophoresis on a sequencing gel. (B) The enzymatic probe used to map the fine structure of the T -2 and T -6 topoisomers is Nuclease SI. Nuclease SI is at 2 mU microL -1 and DNA at 0.5 nM. After the Nuclease SI reaction, the samples are treated to remove the proteins. The DNAs are precipitated and submitted to the BamHI+HindIII double digestion to only visualize DNA fragments from one of the two radiolabeled strands. The reaction products are analyzed on two different sequencing gels (8% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 5) or the guanines and adenines (G+A lanes; lanes 2 and 6). (C) Same as 3B except that the samples are submitted to the BglII+BamHI double digestion to only visualize DNA fragments from the complementary radiolabeled strands. The reaction products are analyzed on two different sequencing gels (7% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 7) or the guanines and adenines (G+A lanes; lanes 2 and 6).

    Techniques Used: Electrophoresis, Sequencing

    11) Product Images from "Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection"

    Article Title: Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0114208

    Lysates of N. gonorrhoeae fragments pECFP-N1 and damage DNA from VK2/E6E7 cells. A. DNA agarose gel showing the digestion of pECFP-N1 plasmid by HindIII (positive control, lane 2), MS11 P+ lysate (lane 3), and MS11 P+ HI lysate (lane 5). Lane 5 shows bacterial MS11 P+ lysate without pECFP-N1 and lane 1 shows uncut circular pECFP-N1. B. PFGE analysis of purified VK2/E6E7 genomic DNA treated for 24 h with: lane 1: PBS (negative control), lane 2: MS11 P+ lysate, lane 3: MS11 P+ HI lysate. Lane 4 shows bacterial MS11 P+ lysate without VK2/E6E7 genomic DNA. C. Graph showing quantification of DNA smears (measured directly underneath and below the band). Shown are smear pixel intensities of cellular DNA alone and cellular DNA exposed to bacterial lysates and HI bacterial lysates. D. PFGE showing genomic DNA subjected to commercial restriction enzymes for 24 h. Lane 1: DNA incubated with CutSmart reaction buffer (negative control). Lane 2: DNA incubated with NgoMIV. Lane 3: DNA incubated with MfeI, Lane 4: DNA incubated with NgoMIV and MfeI Lane 5: DNA incubated with NgoMIV and BamHI/KpnI/MfeI (BKM).
    Figure Legend Snippet: Lysates of N. gonorrhoeae fragments pECFP-N1 and damage DNA from VK2/E6E7 cells. A. DNA agarose gel showing the digestion of pECFP-N1 plasmid by HindIII (positive control, lane 2), MS11 P+ lysate (lane 3), and MS11 P+ HI lysate (lane 5). Lane 5 shows bacterial MS11 P+ lysate without pECFP-N1 and lane 1 shows uncut circular pECFP-N1. B. PFGE analysis of purified VK2/E6E7 genomic DNA treated for 24 h with: lane 1: PBS (negative control), lane 2: MS11 P+ lysate, lane 3: MS11 P+ HI lysate. Lane 4 shows bacterial MS11 P+ lysate without VK2/E6E7 genomic DNA. C. Graph showing quantification of DNA smears (measured directly underneath and below the band). Shown are smear pixel intensities of cellular DNA alone and cellular DNA exposed to bacterial lysates and HI bacterial lysates. D. PFGE showing genomic DNA subjected to commercial restriction enzymes for 24 h. Lane 1: DNA incubated with CutSmart reaction buffer (negative control). Lane 2: DNA incubated with NgoMIV. Lane 3: DNA incubated with MfeI, Lane 4: DNA incubated with NgoMIV and MfeI Lane 5: DNA incubated with NgoMIV and BamHI/KpnI/MfeI (BKM).

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

    12) Product Images from "β-catenin promotes intracellular bacterial killing via suppression of Pseudomonas aeruginosa-triggered macrophage autophagy"

    Article Title: β-catenin promotes intracellular bacterial killing via suppression of Pseudomonas aeruginosa-triggered macrophage autophagy

    Journal: The Journal of International Medical Research

    doi: 10.1177/0300060517692147

    Construction of murine macrophage-like RAW264.7 cells with stable catenin beta 1 expression: (a) Flow cytometry scatter plot showing RAW264.7 cells transduced with control lentivirus or β-catenin lentivirus, sorted according to mCherry marker expression; (b) Representative Western blot showing β-catenin levels in control and β-catenin-transduced monoclones; (c) β-catenin relative integrated density values normalized to β-actin, presented as percentage of control transduced cells (*** P
    Figure Legend Snippet: Construction of murine macrophage-like RAW264.7 cells with stable catenin beta 1 expression: (a) Flow cytometry scatter plot showing RAW264.7 cells transduced with control lentivirus or β-catenin lentivirus, sorted according to mCherry marker expression; (b) Representative Western blot showing β-catenin levels in control and β-catenin-transduced monoclones; (c) β-catenin relative integrated density values normalized to β-actin, presented as percentage of control transduced cells (*** P

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Transduction, Marker, Western Blot

    13) Product Images from "Mesoscopic Liquid Clusters Represent a Distinct Condensate of Mutant p53"

    Article Title: Mesoscopic Liquid Clusters Represent a Distinct Condensate of Mutant p53

    Journal: bioRxiv

    doi: 10.1101/2020.02.04.931980

    Mesoscopic protein-rich clusters of p53 R248Q. a. Schematic of oblique illumination microscopy (OIM). A thin solution is illuminated by green laser at an oblique angle. Scattered light is collected by a microscope lens b. Representative OIM micrographs tracing the evolution of a 2 μM P53 R248Q solution at 15 °C. The observed volume is 5 × 80 × 120 μm 3 depth × height × width. The clusters appear as gold speckles. c. Number density distribution of the radii R of clusters determined by OIM. The averages of five measurements are displayed. The error bars represent the respective standard deviations. d. e. The evolution of the average radius R and number concentration N of clusters determined at 15°C by OIM from images as in b. The averages of five measurements are displayed. The respective standard deviations are smaller than the symbol size. Horizontal lines denote the mean values of R and N . f, g. The concentration dependence of R and N determined at 15°C by OIM. The averages of five measurements are displayed. The error bars represent the respective standard deviations and are smaller than the symbol size for most determinations. Horizontal line in f denotes the mean value of R ; curve in g is a guide to the eye. h, i. The concentration dependence of R and the cluster volume fraction ϕ 2 determined at 15°C by dynamic light scattering. The averages of five measurements are displayed. The error bars represent the respective standard deviations and are smaller than the symbol size for some determinations. Horizontal line in h denotes the mean value of R ; line in g is a guide to the eye.
    Figure Legend Snippet: Mesoscopic protein-rich clusters of p53 R248Q. a. Schematic of oblique illumination microscopy (OIM). A thin solution is illuminated by green laser at an oblique angle. Scattered light is collected by a microscope lens b. Representative OIM micrographs tracing the evolution of a 2 μM P53 R248Q solution at 15 °C. The observed volume is 5 × 80 × 120 μm 3 depth × height × width. The clusters appear as gold speckles. c. Number density distribution of the radii R of clusters determined by OIM. The averages of five measurements are displayed. The error bars represent the respective standard deviations. d. e. The evolution of the average radius R and number concentration N of clusters determined at 15°C by OIM from images as in b. The averages of five measurements are displayed. The respective standard deviations are smaller than the symbol size. Horizontal lines denote the mean values of R and N . f, g. The concentration dependence of R and N determined at 15°C by OIM. The averages of five measurements are displayed. The error bars represent the respective standard deviations and are smaller than the symbol size for most determinations. Horizontal line in f denotes the mean value of R ; curve in g is a guide to the eye. h, i. The concentration dependence of R and the cluster volume fraction ϕ 2 determined at 15°C by dynamic light scattering. The averages of five measurements are displayed. The error bars represent the respective standard deviations and are smaller than the symbol size for some determinations. Horizontal line in h denotes the mean value of R ; line in g is a guide to the eye.

    Techniques Used: Microscopy, Concentration Assay

    Conformational changes induced by the R248Q mutation. a. Comparison of wild type DBD structures corresponding to F ( q ) minima at q = 0.555 (silver) and q = 0.515 (gold). Red box highlights the aggregation-prone sequence, protected by the N-terminus tail in wild type p53 and exposed in p53 R248Q. Green star indicates residues 168 to 193, the location of the strongest deviation of between the two modeled wild type conformations. b. Comparisons of wild type DBD structure corresponding to the F ( q ) minimum at q = 0.555 (silver) to the DBD structures of p53 R248Q (gold) at F ( q ) minima at q = 0.425 in b ; at q = 0.445 in c ; and at q = 0.455 in d . In a – d , the N-terminus tail of the reference structure is highlighted in charcoal, and that of the second structure, in copper. e. Free energy profiles F ( q ) for the cores of the DBDs (residues 107 to 276) of wild type and p53 R248Q.
    Figure Legend Snippet: Conformational changes induced by the R248Q mutation. a. Comparison of wild type DBD structures corresponding to F ( q ) minima at q = 0.555 (silver) and q = 0.515 (gold). Red box highlights the aggregation-prone sequence, protected by the N-terminus tail in wild type p53 and exposed in p53 R248Q. Green star indicates residues 168 to 193, the location of the strongest deviation of between the two modeled wild type conformations. b. Comparisons of wild type DBD structure corresponding to the F ( q ) minimum at q = 0.555 (silver) to the DBD structures of p53 R248Q (gold) at F ( q ) minima at q = 0.425 in b ; at q = 0.445 in c ; and at q = 0.455 in d . In a – d , the N-terminus tail of the reference structure is highlighted in charcoal, and that of the second structure, in copper. e. Free energy profiles F ( q ) for the cores of the DBDs (residues 107 to 276) of wild type and p53 R248Q.

    Techniques Used: Mutagenesis, Sequencing

    Purification and identification of p53 R248Q. a. SDS-PAGE after histidine purification (left), Heparin purification (middle), and buffer exchange (right). b. Absorbance at 280 nm and conductivity of a p53 R248Q solution from heparin resin chromatography column with 2 M NaCl gradient. c. Western blot of p53 R248Q (Primary antibody: DO-1; secondary antibody: m-lgGκ BP-HRP).
    Figure Legend Snippet: Purification and identification of p53 R248Q. a. SDS-PAGE after histidine purification (left), Heparin purification (middle), and buffer exchange (right). b. Absorbance at 280 nm and conductivity of a p53 R248Q solution from heparin resin chromatography column with 2 M NaCl gradient. c. Western blot of p53 R248Q (Primary antibody: DO-1; secondary antibody: m-lgGκ BP-HRP).

    Techniques Used: Purification, SDS Page, Buffer Exchange, Chromatography, Western Blot

    p53 R248Q clusters at temperatures between 15 and 42°C. a. Representative OIM micrographs collected from a p53 R248Q solution with concentration C 0 = 2. μM after incubation for 20 minutes at each temperature. The observed volume is 5 × 80 × 120 μm 3 depth × height × width. Aggregates appear as cyan speckles. b, c. Concentrations C f and C 2 , defined in the plot, of wild type p53, in b , and P53 R248Q, in c , after incubation for 20 min at 15°C as a function of the initial solution concentration C 0 . d, e . The average radius R and the total number N, respectively, of the aggregates, determined by OIM from images as in a . The average of five determinations in distinct solution volumes is shown. Error bars indicate standard deviations and may be smaller than the symbol size. Lines are just guides to the eye.
    Figure Legend Snippet: p53 R248Q clusters at temperatures between 15 and 42°C. a. Representative OIM micrographs collected from a p53 R248Q solution with concentration C 0 = 2. μM after incubation for 20 minutes at each temperature. The observed volume is 5 × 80 × 120 μm 3 depth × height × width. Aggregates appear as cyan speckles. b, c. Concentrations C f and C 2 , defined in the plot, of wild type p53, in b , and P53 R248Q, in c , after incubation for 20 min at 15°C as a function of the initial solution concentration C 0 . d, e . The average radius R and the total number N, respectively, of the aggregates, determined by OIM from images as in a . The average of five determinations in distinct solution volumes is shown. Error bars indicate standard deviations and may be smaller than the symbol size. Lines are just guides to the eye.

    Techniques Used: Concentration Assay, Incubation

    Aggregated p53 R248Q in a breast cancer HCC70 cell. Combined staining with Pab240, which binds to unfolded or aggregated p53, and ThT, which detects amyloid structures. The cell nucleus is stained with a Hoechst dye. A confocal image of a representative single cell is shown.
    Figure Legend Snippet: Aggregated p53 R248Q in a breast cancer HCC70 cell. Combined staining with Pab240, which binds to unfolded or aggregated p53, and ThT, which detects amyloid structures. The cell nucleus is stained with a Hoechst dye. A confocal image of a representative single cell is shown.

    Techniques Used: Staining

    The structure of unaggregated p53 R248Q. a. Normalized intensity correlation functions g 2 (τ) of the light scattered by filtered p53 R248Q solutions at shown concentrations at 15°C. The characteristic diffusion times of unaggregated protein τ 1 and of the τ 2 clusters τ 2 are shown. b, c. Average radius R m and dispersity μτ 2 of unaggregated p53 R248Q determined from the g 2 s in a. for p53 R248Q. Data for wild type p53 38 are shown for comparison. d. The osmotic compressibility of the solution KC / R θ ( K , instrument constant; R θ , Rayleigh ratio of the intensity of light scattered at 90° to the incident light; C, concentration of p53) as a function of concentration for wild type p53 at 15 °C and R248Q at 15 °C, 24 °C, and 37 °C. e. The average molecular weight of unaggregated p53 R248Q evaluated form the KC / R θ ( C ) data in d at three temperatures.
    Figure Legend Snippet: The structure of unaggregated p53 R248Q. a. Normalized intensity correlation functions g 2 (τ) of the light scattered by filtered p53 R248Q solutions at shown concentrations at 15°C. The characteristic diffusion times of unaggregated protein τ 1 and of the τ 2 clusters τ 2 are shown. b, c. Average radius R m and dispersity μτ 2 of unaggregated p53 R248Q determined from the g 2 s in a. for p53 R248Q. Data for wild type p53 38 are shown for comparison. d. The osmotic compressibility of the solution KC / R θ ( K , instrument constant; R θ , Rayleigh ratio of the intensity of light scattered at 90° to the incident light; C, concentration of p53) as a function of concentration for wild type p53 at 15 °C and R248Q at 15 °C, 24 °C, and 37 °C. e. The average molecular weight of unaggregated p53 R248Q evaluated form the KC / R θ ( C ) data in d at three temperatures.

    Techniques Used: Diffusion-based Assay, Concentration Assay, Molecular Weight

    The R248Q mutation and aggregation on p53 R248Q in breast cancer cells. a. The structure of the DNA-binding domain (DBD, 94-292) of wild-type p53 and the R248Q (Arg 248 Gln) mutant. Nitrogen atoms are colored in red, zinc in green, alpha-helices in purple, beta sheets in orange, and DNA in blue. PDB ID: 1TUP 40 for wild type p53; prediction of p53 R248Q DNA interaction using VMD. 41 b. Schematic of confocal immunofluorescent microcopy, in which antibodies that specifically target cell components of interest are tagged with fluorescent dyes. The spatial distribution of the fluorescent signal collected by the microscope maps the 3D distribution of the target molecule. c. Illustration of diffuse and punctate staining in the cytoplasm. d. Combined staining with Pab240, which binds to unfolded or aggregated p53, and ThT, which detects amyloid structures. The cell nucleus is stained with a Hoechst dye. e. Staining of HCC70 cells with Pab240 before and after treatment with 1,6-hexanediol, known to disperse dense liquid droplets of disordered proteins. f. Distributions of the volumes of the puncta found in HCC70 cells treated with Pab240. Each trace represents the volume distribution of puncta from a single cell.
    Figure Legend Snippet: The R248Q mutation and aggregation on p53 R248Q in breast cancer cells. a. The structure of the DNA-binding domain (DBD, 94-292) of wild-type p53 and the R248Q (Arg 248 Gln) mutant. Nitrogen atoms are colored in red, zinc in green, alpha-helices in purple, beta sheets in orange, and DNA in blue. PDB ID: 1TUP 40 for wild type p53; prediction of p53 R248Q DNA interaction using VMD. 41 b. Schematic of confocal immunofluorescent microcopy, in which antibodies that specifically target cell components of interest are tagged with fluorescent dyes. The spatial distribution of the fluorescent signal collected by the microscope maps the 3D distribution of the target molecule. c. Illustration of diffuse and punctate staining in the cytoplasm. d. Combined staining with Pab240, which binds to unfolded or aggregated p53, and ThT, which detects amyloid structures. The cell nucleus is stained with a Hoechst dye. e. Staining of HCC70 cells with Pab240 before and after treatment with 1,6-hexanediol, known to disperse dense liquid droplets of disordered proteins. f. Distributions of the volumes of the puncta found in HCC70 cells treated with Pab240. Each trace represents the volume distribution of puncta from a single cell.

    Techniques Used: Mutagenesis, Binding Assay, Microscopy, Staining

    Unaggregated wild type p53 and p53 R248Q in breast cancer cells. a,b. Combined staining with Hoechst, which stains the nucleus, and DO1, which binds to unaggregated p53. Confocal images of a. A representative HCC70 single-cell. b. A repsreeitnative MCF7 cell.
    Figure Legend Snippet: Unaggregated wild type p53 and p53 R248Q in breast cancer cells. a,b. Combined staining with Hoechst, which stains the nucleus, and DO1, which binds to unaggregated p53. Confocal images of a. A representative HCC70 single-cell. b. A repsreeitnative MCF7 cell.

    Techniques Used: Staining

    Fibrillization of wild type and p53 R248Q. a – d. Evolution of the intensity of fluorescence at 500 nm of 1-anilino-8-naphthalenesulfonate (ANS) in the presence of p53 at 37°C. ANS concentrations was 200 μM in all tests. a, b. At the listed concentrations of wild type, in a , and R248Q, in b , in the absence of Ficoll. c, d. At 6.5 μM of wild type, in c , and R248Q, in d , and in the presence of varying concentrations of Ficoll. e. Schematic of two-step nucleation of fibrils. Step 1. Mesoscopic p53-rich clusters form from misassembled p53 oligomers and native tetramers. Step 2. Fibrils nucleate within the mesoscopic clusters. Fibril growth proceeds classically, via sequential association of p53 monomers from the solution.
    Figure Legend Snippet: Fibrillization of wild type and p53 R248Q. a – d. Evolution of the intensity of fluorescence at 500 nm of 1-anilino-8-naphthalenesulfonate (ANS) in the presence of p53 at 37°C. ANS concentrations was 200 μM in all tests. a, b. At the listed concentrations of wild type, in a , and R248Q, in b , in the absence of Ficoll. c, d. At 6.5 μM of wild type, in c , and R248Q, in d , and in the presence of varying concentrations of Ficoll. e. Schematic of two-step nucleation of fibrils. Step 1. Mesoscopic p53-rich clusters form from misassembled p53 oligomers and native tetramers. Step 2. Fibrils nucleate within the mesoscopic clusters. Fibril growth proceeds classically, via sequential association of p53 monomers from the solution.

    Techniques Used: Fluorescence

    The free energy F of the conformations of wild type and p53 R248Q DNA binding domains. a. As a function of the reaction coordinate q that measures the similarity of the DBD conformation to a structure determined by x-ray crystallography. 40 . b – e. Two dimensional profiles of F as function of q and radius of gyration, R g , in b and c , and end-to-end distance, D , in d and e , of the DBD chain
    Figure Legend Snippet: The free energy F of the conformations of wild type and p53 R248Q DNA binding domains. a. As a function of the reaction coordinate q that measures the similarity of the DBD conformation to a structure determined by x-ray crystallography. 40 . b – e. Two dimensional profiles of F as function of q and radius of gyration, R g , in b and c , and end-to-end distance, D , in d and e , of the DBD chain

    Techniques Used: Binding Assay

    14) Product Images from "Genetic Stabilization of the Drug-Resistant PMEN1 Pneumococcus Lineage by Its Distinctive DpnIII Restriction-Modification System"

    Article Title: Genetic Stabilization of the Drug-Resistant PMEN1 Pneumococcus Lineage by Its Distinctive DpnIII Restriction-Modification System

    Journal: mBio

    doi: 10.1128/mBio.00173-15

    Characterization of (R-M system) DpnIII demonstrating that R.DpnIII cleaves DNA at 5′ GATC 3′ and M.DpnIII methylates DNA at the cytosine. (A) Digestion of pUC19 and spectinomycin R with a histidine-tagged DpnIII-enriched fraction and Sau3AI, showing bands consistent with digestion at GATC. (B) Genomic DNA isolated from the WT and RMKO strains combined with endonucleases that cleave at GATC but are inhibited by methylation at different positions (cleavage by BamHI, BglII, and Sau3AI is inhibited by methylation of the cytosine, and cleavage by BclI and MboI is inhibited by methylation of the adenine). (C) WT and RMKO DNA mixed with Sau3AI and histidine-tagged DpnIII, where only the RMKO is susceptible to digestion. Further, WT DNA of strain 8140 is protected by digestion with Sau3AI and DpnIII. Enz., enzyme; MM, mass markers. The values to the left of panel A are molecular masses in base pairs.
    Figure Legend Snippet: Characterization of (R-M system) DpnIII demonstrating that R.DpnIII cleaves DNA at 5′ GATC 3′ and M.DpnIII methylates DNA at the cytosine. (A) Digestion of pUC19 and spectinomycin R with a histidine-tagged DpnIII-enriched fraction and Sau3AI, showing bands consistent with digestion at GATC. (B) Genomic DNA isolated from the WT and RMKO strains combined with endonucleases that cleave at GATC but are inhibited by methylation at different positions (cleavage by BamHI, BglII, and Sau3AI is inhibited by methylation of the cytosine, and cleavage by BclI and MboI is inhibited by methylation of the adenine). (C) WT and RMKO DNA mixed with Sau3AI and histidine-tagged DpnIII, where only the RMKO is susceptible to digestion. Further, WT DNA of strain 8140 is protected by digestion with Sau3AI and DpnIII. Enz., enzyme; MM, mass markers. The values to the left of panel A are molecular masses in base pairs.

    Techniques Used: Isolation, Methylation

    15) Product Images from "Evidence Suggesting Absence of Mitochondrial DNA Methylation"

    Article Title: Evidence Suggesting Absence of Mitochondrial DNA Methylation

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2017.00166

    BamHI digestion prior to bisulfite sequencing decreases cytosine unconvertion rate. Targeted bisulfite sequencing was used to compare undigested and digested DNA methylation levels at five different regions of the mtDNA from human muscle cells and SKOV3 cells ( N = 3). (A) Drawing displays the mtDNA regions investigated by targeted bisulfite sequencing. (B) Percentage methylation for undigested and digested mtDNA. Full circle represents cytosines in CpG context whereas open circle is cytosines in non-CpG context. Results are presented with a min-max interval and a sign test was used to test for significant methylation differences. D-loop (6–298) P = 2.02E-41 (Lonza) and P = 1.40E-24 (SKOV3); D-loop (279–458): P = 5.00E-12 (Lonza) and P = 8.20E-08 (SKOV3); tRNA-F+12S: P = 9.22E-15 (Lonza) and P = 1.29E-9 (SKOV3); 16S: P = 6.50E-05; ND5: P = 1.70E-05 (Lonza) and P = 9.97E-13 (SKOV3); CYTB: P = 2.96E-09 (Lonza), and P = 6.68E-13 (SKOV3). D-loop (6–298) includes origin of replication and tRNA-F+12S includes heavy strand promoter 2. P H1 : heavy-strand promoter 1; P H2 : Heavy-strand promote 2; P L : Light-strand promoter; OH: Origin of replication from heavy-strand; O L : Origin of replication from light-strand. ND: not determined.
    Figure Legend Snippet: BamHI digestion prior to bisulfite sequencing decreases cytosine unconvertion rate. Targeted bisulfite sequencing was used to compare undigested and digested DNA methylation levels at five different regions of the mtDNA from human muscle cells and SKOV3 cells ( N = 3). (A) Drawing displays the mtDNA regions investigated by targeted bisulfite sequencing. (B) Percentage methylation for undigested and digested mtDNA. Full circle represents cytosines in CpG context whereas open circle is cytosines in non-CpG context. Results are presented with a min-max interval and a sign test was used to test for significant methylation differences. D-loop (6–298) P = 2.02E-41 (Lonza) and P = 1.40E-24 (SKOV3); D-loop (279–458): P = 5.00E-12 (Lonza) and P = 8.20E-08 (SKOV3); tRNA-F+12S: P = 9.22E-15 (Lonza) and P = 1.29E-9 (SKOV3); 16S: P = 6.50E-05; ND5: P = 1.70E-05 (Lonza) and P = 9.97E-13 (SKOV3); CYTB: P = 2.96E-09 (Lonza), and P = 6.68E-13 (SKOV3). D-loop (6–298) includes origin of replication and tRNA-F+12S includes heavy strand promoter 2. P H1 : heavy-strand promoter 1; P H2 : Heavy-strand promote 2; P L : Light-strand promoter; OH: Origin of replication from heavy-strand; O L : Origin of replication from light-strand. ND: not determined.

    Techniques Used: Methylation Sequencing, DNA Methylation Assay, Methylation

    16) Product Images from "Genetic Stabilization of the Drug-Resistant PMEN1 Pneumococcus Lineage by Its Distinctive DpnIII Restriction-Modification System"

    Article Title: Genetic Stabilization of the Drug-Resistant PMEN1 Pneumococcus Lineage by Its Distinctive DpnIII Restriction-Modification System

    Journal: mBio

    doi: 10.1128/mBio.00173-15

    Characterization of (R-M system) DpnIII demonstrating that R.DpnIII cleaves DNA at 5′ GATC 3′ and M.DpnIII methylates DNA at the cytosine. (A) Digestion of pUC19 and spectinomycin R with a histidine-tagged DpnIII-enriched fraction and Sau3AI, showing bands consistent with digestion at GATC. (B) Genomic DNA isolated from the WT and RMKO strains combined with endonucleases that cleave at GATC but are inhibited by methylation at different positions (cleavage by BamHI, BglII, and Sau3AI is inhibited by methylation of the cytosine, and cleavage by BclI and MboI is inhibited by methylation of the adenine). (C) WT and RMKO DNA mixed with Sau3AI and histidine-tagged DpnIII, where only the RMKO is susceptible to digestion. Further, WT DNA of strain 8140 is protected by digestion with Sau3AI and DpnIII. Enz., enzyme; MM, mass markers. The values to the left of panel A are molecular masses in base pairs.
    Figure Legend Snippet: Characterization of (R-M system) DpnIII demonstrating that R.DpnIII cleaves DNA at 5′ GATC 3′ and M.DpnIII methylates DNA at the cytosine. (A) Digestion of pUC19 and spectinomycin R with a histidine-tagged DpnIII-enriched fraction and Sau3AI, showing bands consistent with digestion at GATC. (B) Genomic DNA isolated from the WT and RMKO strains combined with endonucleases that cleave at GATC but are inhibited by methylation at different positions (cleavage by BamHI, BglII, and Sau3AI is inhibited by methylation of the cytosine, and cleavage by BclI and MboI is inhibited by methylation of the adenine). (C) WT and RMKO DNA mixed with Sau3AI and histidine-tagged DpnIII, where only the RMKO is susceptible to digestion. Further, WT DNA of strain 8140 is protected by digestion with Sau3AI and DpnIII. Enz., enzyme; MM, mass markers. The values to the left of panel A are molecular masses in base pairs.

    Techniques Used: Isolation, Methylation

    17) Product Images from "Enhanced antifungal activity of bovine lactoferrin-producing probiotic Lactobacillus casei in the murine model of vulvovaginal candidiasis"

    Article Title: Enhanced antifungal activity of bovine lactoferrin-producing probiotic Lactobacillus casei in the murine model of vulvovaginal candidiasis

    Journal: BMC Microbiology

    doi: 10.1186/s12866-018-1370-x

    Construction and expression of the secretion plasmid pPG612.1-BLF in L.casei . a The synthetic BLF gene fragment (2.1kp) was digested with restriction enzymes BamHI and XhoI, and ligated into the sticky end of the plasmid pPG612.1 which was also digested with the same restriction enzyme, resulting in the plasmid pPG612.1-BLF (5.6kp). b The plasmid pPG612.1-BLF was electroporated into L.casei using a BioRad GenePulser with single electric pulse (voltage, 1.5 kV; capacitance, 25 μF; and resistance, 400 Ω.). PCR amplification of the BamHI site and XhoI site of the plasmid pPG612.1-BLF which was extracted from the L.casei /pPG612.1-BLF strain resulted in 500 bp and 800 bp products, respectively. Lane 1, PCR product of XhoI site; Lane 2, PCR product of BamHI site. M, DNA maker. c BLF was detected in the supernatant and pellet of L.casei /pPG612.1-BLF culture by Western blotting, indicating the expression and secretion of BLF by L.casei /pPG612.1-BLF. Lane 1, supernatant of L.casei /pPG612.1-BLF culture; Lane 2, pellet of L.casei /pPG612.1-BLF culture; Lane 3, supernatant of L.casei /pPG612.1 culture; Lane 4, pellet of L.casei /pPG612.1 culture
    Figure Legend Snippet: Construction and expression of the secretion plasmid pPG612.1-BLF in L.casei . a The synthetic BLF gene fragment (2.1kp) was digested with restriction enzymes BamHI and XhoI, and ligated into the sticky end of the plasmid pPG612.1 which was also digested with the same restriction enzyme, resulting in the plasmid pPG612.1-BLF (5.6kp). b The plasmid pPG612.1-BLF was electroporated into L.casei using a BioRad GenePulser with single electric pulse (voltage, 1.5 kV; capacitance, 25 μF; and resistance, 400 Ω.). PCR amplification of the BamHI site and XhoI site of the plasmid pPG612.1-BLF which was extracted from the L.casei /pPG612.1-BLF strain resulted in 500 bp and 800 bp products, respectively. Lane 1, PCR product of XhoI site; Lane 2, PCR product of BamHI site. M, DNA maker. c BLF was detected in the supernatant and pellet of L.casei /pPG612.1-BLF culture by Western blotting, indicating the expression and secretion of BLF by L.casei /pPG612.1-BLF. Lane 1, supernatant of L.casei /pPG612.1-BLF culture; Lane 2, pellet of L.casei /pPG612.1-BLF culture; Lane 3, supernatant of L.casei /pPG612.1 culture; Lane 4, pellet of L.casei /pPG612.1 culture

    Techniques Used: Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Western Blot

    18) Product Images from "Resistance to 6-Methylpurine is Conferred by Defective Adenine Phosphoribosyltransferase in Tetrahymena"

    Article Title: Resistance to 6-Methylpurine is Conferred by Defective Adenine Phosphoribosyltransferase in Tetrahymena

    Journal: Genes

    doi: 10.3390/genes9040179

    Effect on cell sensitivity to 6mp of replacing wild-type APRT1 with the mutant gene. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pD127N-FZZ-PAC, with the mutant gene, FZZ tag containing polyA signal, and puromycin resistant cassette ( PAC ) (middle), and after homologous recombination (lower). Control plasmid carries wild-type APRT1 instead of the mutated version. ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and FZZ-tagged APRTase-expressing transformants. The molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Western blot analysis of FZZ-tagged APRTases. FZZ tag and APRTase were 17 kDa and 20 kDa, respectively, resulting in a single 37 kDa band. Tubulin-alpha was the loading control. ( D ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h.
    Figure Legend Snippet: Effect on cell sensitivity to 6mp of replacing wild-type APRT1 with the mutant gene. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pD127N-FZZ-PAC, with the mutant gene, FZZ tag containing polyA signal, and puromycin resistant cassette ( PAC ) (middle), and after homologous recombination (lower). Control plasmid carries wild-type APRT1 instead of the mutated version. ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and FZZ-tagged APRTase-expressing transformants. The molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Western blot analysis of FZZ-tagged APRTases. FZZ tag and APRTase were 17 kDa and 20 kDa, respectively, resulting in a single 37 kDa band. Tubulin-alpha was the loading control. ( D ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h.

    Techniques Used: Mutagenesis, Plasmid Preparation, Homologous Recombination, Southern Blot, Expressing, Molecular Weight, Western Blot

    Effect of partial APRT1 knockout on cell sensitivity to 6mp. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pΔAPRT1-NEO5, with paromomycin resistance cassette ( NEO5 ) (middle), and after homologous recombination (lower). ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and partial APRT1 knockout cells. Molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h. ( D ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after measurement of cell growth rates in 6mp. ( E ) Southern blot analysis of XhoI-and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after the induction of phenotypic assortment with 5 mg/mL paromomycin for 2 months. Molecular weight of signals against the probe corresponds to that predicted in the schematic in ( A ).
    Figure Legend Snippet: Effect of partial APRT1 knockout on cell sensitivity to 6mp. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pΔAPRT1-NEO5, with paromomycin resistance cassette ( NEO5 ) (middle), and after homologous recombination (lower). ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and partial APRT1 knockout cells. Molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h. ( D ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after measurement of cell growth rates in 6mp. ( E ) Southern blot analysis of XhoI-and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after the induction of phenotypic assortment with 5 mg/mL paromomycin for 2 months. Molecular weight of signals against the probe corresponds to that predicted in the schematic in ( A ).

    Techniques Used: Knock-Out, Plasmid Preparation, Homologous Recombination, Southern Blot, Molecular Weight

    19) Product Images from "PhaQ, a New Class of Poly-?-Hydroxybutyrate (PHB)-Responsive Repressor, Regulates phaQ and phaP (Phasin) Expression in Bacillus megaterium through Interaction with PHB"

    Article Title: PhaQ, a New Class of Poly-?-Hydroxybutyrate (PHB)-Responsive Repressor, Regulates phaQ and phaP (Phasin) Expression in Bacillus megaterium through Interaction with PHB

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.186.10.3015-3021.2004

    DNase I footprinting analysis of PhaQ binding to the phaQ promoter region. (A) A 0.4-kb SmaI-HindIII DNA fragment containing the phaQ promoter region (positions −356 to +39) and labeled with 32 P at its HindIII site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. (B) A 0.36-kb BamHI-EcoRI DNA fragment containing the phaQ promoter region (positions −105 to + 249) and labeled with 32 P at its BamHI site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. The numbers on the left indicate the positions of bases relative to the transcriptional initiation site of phaQ . Solid brackets on the right denote the protected regions.
    Figure Legend Snippet: DNase I footprinting analysis of PhaQ binding to the phaQ promoter region. (A) A 0.4-kb SmaI-HindIII DNA fragment containing the phaQ promoter region (positions −356 to +39) and labeled with 32 P at its HindIII site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. (B) A 0.36-kb BamHI-EcoRI DNA fragment containing the phaQ promoter region (positions −105 to + 249) and labeled with 32 P at its BamHI site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. The numbers on the left indicate the positions of bases relative to the transcriptional initiation site of phaQ . Solid brackets on the right denote the protected regions.

    Techniques Used: Footprinting, Binding Assay, Labeling, Incubation

    DNase I footprinting analysis of PhaQ binding to the phaQ promoter region. (A) A 0.4-kb SmaI-HindIII DNA fragment containing the phaQ promoter region (positions −356 to +39) and labeled with 32 P at its HindIII site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. (B) A 0.36-kb BamHI-EcoRI DNA fragment containing the phaQ promoter region (positions −105 to + 249) and labeled with 32 P at its BamHI site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. The numbers on the left indicate the positions of bases relative to the transcriptional initiation site of phaQ . Solid brackets on the right denote the protected regions.
    Figure Legend Snippet: DNase I footprinting analysis of PhaQ binding to the phaQ promoter region. (A) A 0.4-kb SmaI-HindIII DNA fragment containing the phaQ promoter region (positions −356 to +39) and labeled with 32 P at its HindIII site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. (B) A 0.36-kb BamHI-EcoRI DNA fragment containing the phaQ promoter region (positions −105 to + 249) and labeled with 32 P at its BamHI site was incubated in the absence or presence of the PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. The numbers on the left indicate the positions of bases relative to the transcriptional initiation site of phaQ . Solid brackets on the right denote the protected regions.

    Techniques Used: Footprinting, Binding Assay, Labeling, Incubation

    20) Product Images from "Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation"

    Article Title: Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

    Journal: bioRxiv

    doi: 10.1101/2020.07.02.184408

    Generation of synthetic α 21-I TetO and α 21-II LacO/Gal4 arrays. (A) Scheme of the pBAC11.32TW12.32GLII containing BAC and YAC cassettes, G418 resistance cassette and synthetic DNA: α 21-I TetO formed by high ordered repeats (HOR) monomers (green arrows) containing CENP-B boxes (blue) alternate with monomers containing TetO (yellow); α 21-II LacO/Gal4 formed by high ordered repeats (HOR) monomers (yellow arrows) containing Gal4 binding sequence (green) alternating with LacO (red). (B) Schematic of the assembly of the α 21-I TetO and α 21-II LacO/Gal4 arrays. (C, D) PFGE analysis of the nascent α 21-I TetO and α 21-II LacO/Gal4 arrays, cut with BamHI/NotI after each cycles of tandem ligation array amplification as described in Figure S2A (C) and Figure S2B (D). Expected sizes: α 21-I TetO 11-mer 1 copy (1.9 kb), 8 copies (15.2 kb), 32 copies (60.8 kb); α 21-II LacO/Gal4 12-mer 1 copy (2 kb), 8 copies (16 kb), 32 copies (64 kb). Plasmid vector is 2.9 kb, BAC vector is 7.1 kb. The asterisk (*) indicates the fragments that have been cloned into BAC vector (8 copies, 16 kb); red arrow in D indicates the size of the final pBAC11.32TW12.32GLII (∼120 kb) (m and M, markers).
    Figure Legend Snippet: Generation of synthetic α 21-I TetO and α 21-II LacO/Gal4 arrays. (A) Scheme of the pBAC11.32TW12.32GLII containing BAC and YAC cassettes, G418 resistance cassette and synthetic DNA: α 21-I TetO formed by high ordered repeats (HOR) monomers (green arrows) containing CENP-B boxes (blue) alternate with monomers containing TetO (yellow); α 21-II LacO/Gal4 formed by high ordered repeats (HOR) monomers (yellow arrows) containing Gal4 binding sequence (green) alternating with LacO (red). (B) Schematic of the assembly of the α 21-I TetO and α 21-II LacO/Gal4 arrays. (C, D) PFGE analysis of the nascent α 21-I TetO and α 21-II LacO/Gal4 arrays, cut with BamHI/NotI after each cycles of tandem ligation array amplification as described in Figure S2A (C) and Figure S2B (D). Expected sizes: α 21-I TetO 11-mer 1 copy (1.9 kb), 8 copies (15.2 kb), 32 copies (60.8 kb); α 21-II LacO/Gal4 12-mer 1 copy (2 kb), 8 copies (16 kb), 32 copies (64 kb). Plasmid vector is 2.9 kb, BAC vector is 7.1 kb. The asterisk (*) indicates the fragments that have been cloned into BAC vector (8 copies, 16 kb); red arrow in D indicates the size of the final pBAC11.32TW12.32GLII (∼120 kb) (m and M, markers).

    Techniques Used: BAC Assay, Binding Assay, Sequencing, Ligation, Amplification, Plasmid Preparation, Clone Assay

    Analysis of rearrangements of the pBAC11.32TW12.32GLII arrays. Timeline of the experiments performed from transfection into HT1080 clones and subclones. (A,B,C) Southern blot of subclones from clone E30 (A), J34 (B) and E16 (C): DNA was digested with BamHI and separated by CHEF. The transferred membrane was hybridized with radioactively labelled TetO (left) or LacO (right) specific probes. Cartoons on the right represent the outcome of rearrangements in the corresponding clone: green arrows represent α 21-I TetO array, yellow arrows represent α 21-II LacO/Gal4 array, boxes represent the BAC vector (M and m, markers).
    Figure Legend Snippet: Analysis of rearrangements of the pBAC11.32TW12.32GLII arrays. Timeline of the experiments performed from transfection into HT1080 clones and subclones. (A,B,C) Southern blot of subclones from clone E30 (A), J34 (B) and E16 (C): DNA was digested with BamHI and separated by CHEF. The transferred membrane was hybridized with radioactively labelled TetO (left) or LacO (right) specific probes. Cartoons on the right represent the outcome of rearrangements in the corresponding clone: green arrows represent α 21-I TetO array, yellow arrows represent α 21-II LacO/Gal4 array, boxes represent the BAC vector (M and m, markers).

    Techniques Used: Transfection, Clone Assay, Southern Blot, BAC Assay, Plasmid Preparation

    Screening of HT1080 colonies after transfection with pBAC11.32TW12.32GLII. (A) Scheme showing the possible fates of the pBAC11.32TW12.32GLII HAC seeding DNA after transfection in HT1080: in yellow and green (as integration or HAC) is represented the synthetic DNA. Timeline of the experiments performed from transfection into HT1080 cells. (B) BAC copy number (y axis) analyzed by qPCR in each HT1080 clone (x axis): only HT1080 clones containing > 20 BAC copies are represented in the graph. HT1080 clones are represented in green (HAC), red (integration) or mixture (both) according to the results of the FISH screening, as shown in C. Black arrows indicate the clones shown in C and analyzed further. (C) Representative pictures of oligo-FISH staining of HT1080 clones: slides have been hybridized with DNA probes (TetO-dig/rhodamine -dig antibody, Gal4-biotin and LacO-biotin/Fitc-streptavidin). DAPI stains DNA. Scalebar = 10µm. (D) Southern blot of selected HT1080 clonal DNA (as labelled on top of the panel) digested with BamHI and separated by CHEF; the transferred membrane was hybridized with radioactively labelled TetO (left) or LacO (right) specific probes. Red arrows indicate the expected size of the band without rearrangements. Clones labelled in red have been screened further (M and m, markers). (E) Cartoon of the pBAC11.32TW12.32GLII input DNA showing restriction sites for NotI and BamHI.
    Figure Legend Snippet: Screening of HT1080 colonies after transfection with pBAC11.32TW12.32GLII. (A) Scheme showing the possible fates of the pBAC11.32TW12.32GLII HAC seeding DNA after transfection in HT1080: in yellow and green (as integration or HAC) is represented the synthetic DNA. Timeline of the experiments performed from transfection into HT1080 cells. (B) BAC copy number (y axis) analyzed by qPCR in each HT1080 clone (x axis): only HT1080 clones containing > 20 BAC copies are represented in the graph. HT1080 clones are represented in green (HAC), red (integration) or mixture (both) according to the results of the FISH screening, as shown in C. Black arrows indicate the clones shown in C and analyzed further. (C) Representative pictures of oligo-FISH staining of HT1080 clones: slides have been hybridized with DNA probes (TetO-dig/rhodamine -dig antibody, Gal4-biotin and LacO-biotin/Fitc-streptavidin). DAPI stains DNA. Scalebar = 10µm. (D) Southern blot of selected HT1080 clonal DNA (as labelled on top of the panel) digested with BamHI and separated by CHEF; the transferred membrane was hybridized with radioactively labelled TetO (left) or LacO (right) specific probes. Red arrows indicate the expected size of the band without rearrangements. Clones labelled in red have been screened further (M and m, markers). (E) Cartoon of the pBAC11.32TW12.32GLII input DNA showing restriction sites for NotI and BamHI.

    Techniques Used: Transfection, HAC Assay, BAC Assay, Real-time Polymerase Chain Reaction, Clone Assay, Fluorescence In Situ Hybridization, Staining, Southern Blot

    Formation of input pBAC11.32TW12.32GLII DNA. (A) CHEF analysis of 16 bacterial DNA after transformation with pBAC11.32TW12.32GLII and NotI and BamHI digestion: red arrows indicate the size of the final vector (∼120 kb); colonies labelled in red contain the insert of the desired length. DNA used for transfection as a control (in duplicate) (M marker). (B) Scheme of the pBAC11.32TW12.32GLII input DNA showing restriction sites for NotI and BamHI used to release the synthetic DNA. (C) PFGE analysis of selected bacterial colonies (in red) digested with EcoRI: each fragment originates from a different array (label on the left). DNA used for transfection as a control (in duplicate); original DNA as uncut sample (M marker). (D) α 21-I TetO and α 21-II LacO/Gal4 DNA ratio calculated with ImageJ on the intensity of the bands shown in C for each bacterial colony. Control and original DNA as in C. (E) CHEF analysis of bacterial colony #1 DNA (in duplicate) digested with NotI and BamHI to release the synthetic DNA (m and M, markers).
    Figure Legend Snippet: Formation of input pBAC11.32TW12.32GLII DNA. (A) CHEF analysis of 16 bacterial DNA after transformation with pBAC11.32TW12.32GLII and NotI and BamHI digestion: red arrows indicate the size of the final vector (∼120 kb); colonies labelled in red contain the insert of the desired length. DNA used for transfection as a control (in duplicate) (M marker). (B) Scheme of the pBAC11.32TW12.32GLII input DNA showing restriction sites for NotI and BamHI used to release the synthetic DNA. (C) PFGE analysis of selected bacterial colonies (in red) digested with EcoRI: each fragment originates from a different array (label on the left). DNA used for transfection as a control (in duplicate); original DNA as uncut sample (M marker). (D) α 21-I TetO and α 21-II LacO/Gal4 DNA ratio calculated with ImageJ on the intensity of the bands shown in C for each bacterial colony. Control and original DNA as in C. (E) CHEF analysis of bacterial colony #1 DNA (in duplicate) digested with NotI and BamHI to release the synthetic DNA (m and M, markers).

    Techniques Used: Transformation Assay, Plasmid Preparation, Transfection, Marker

    21) Product Images from "Loss of a doublecortin (DCX) domain containing protein causes structural defects in a tubulin-based organelle of Toxoplasma gondii and impairs host cell invasion"

    Article Title: Loss of a doublecortin (DCX) domain containing protein causes structural defects in a tubulin-based organelle of Toxoplasma gondii and impairs host cell invasion

    Journal: bioRxiv

    doi: 10.1101/069377

    Southern blot with T. gondii genomic DNA. Left side: Map of the TgDCX genomic region in parental (RHΔHXΔKu80), knock-in (FP-TgDCX or TgDCX-FP), and knockout (ΔTgDCX) parasites. The sizes of the BamHI-EcoRV or EcoRV-EcoRV fragments containing the hybridization targets are indicated. Sequence regions included in the probes are indicated by the striped regions; exon 2–5 probe, grey-white stripes in knock-in map; 5’-UTR probe, grey-black stripes in the knockout map. The 5’-UTR region is identical in the three parasite genomes. Right side: size marker (length in kbp); Lanes 1–5: hybridized with a probe against exons 2–5 of TgDCX CDS. lane 1, RHΔHXΔKu80 parasites, expected size 4412 bp; lane 2 and 3, mCherryFP-TgDCX and TgDCX-NeonGreenFP knock-in parasites, expected size 3620 bp; lane 4 and 5, clones of knockout parasites derived from mCherryFP-TgDCX and TgDCX-NeonGreenFP knock-in parasites respectively, no band expected. Lanes 4a and 5a are lanes 4 and 5 respectively of the original blot after it was stripped and re-hybridized with a probe against the 5’-UTR region of the TgDCX gene. expected size 2926 bp.
    Figure Legend Snippet: Southern blot with T. gondii genomic DNA. Left side: Map of the TgDCX genomic region in parental (RHΔHXΔKu80), knock-in (FP-TgDCX or TgDCX-FP), and knockout (ΔTgDCX) parasites. The sizes of the BamHI-EcoRV or EcoRV-EcoRV fragments containing the hybridization targets are indicated. Sequence regions included in the probes are indicated by the striped regions; exon 2–5 probe, grey-white stripes in knock-in map; 5’-UTR probe, grey-black stripes in the knockout map. The 5’-UTR region is identical in the three parasite genomes. Right side: size marker (length in kbp); Lanes 1–5: hybridized with a probe against exons 2–5 of TgDCX CDS. lane 1, RHΔHXΔKu80 parasites, expected size 4412 bp; lane 2 and 3, mCherryFP-TgDCX and TgDCX-NeonGreenFP knock-in parasites, expected size 3620 bp; lane 4 and 5, clones of knockout parasites derived from mCherryFP-TgDCX and TgDCX-NeonGreenFP knock-in parasites respectively, no band expected. Lanes 4a and 5a are lanes 4 and 5 respectively of the original blot after it was stripped and re-hybridized with a probe against the 5’-UTR region of the TgDCX gene. expected size 2926 bp.

    Techniques Used: Southern Blot, Knock-In, Knock-Out, Hybridization, Sequencing, Marker, Clone Assay, Derivative Assay

    22) Product Images from "Direct detection of methylation in genomic DNA"

    Article Title: Direct detection of methylation in genomic DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni121

    Differences between G signals complementary to unmethylated C residues, N 4 -methylcytosine residues and 5-methylcytosine residues using the DYEnamic ET Terminator Kit. A1: untreated pUC19 DNA; A2: M.BamHI N 4 -cytosine methylated pUC19DNA; A3: trace difference between A2 and A1. B1: untreated pUC19DNA; B2: M.HaeIII 5-cytosine methylated pUC19 DNA; B3: trace difference between B2 and B1. The G residues complementary to the methylated cytosines are boxed. N 4 -methylcytosine results in an increase in the complementary G signal, whereas 5-methylcytosine results in a decrease in the complementary G signal.
    Figure Legend Snippet: Differences between G signals complementary to unmethylated C residues, N 4 -methylcytosine residues and 5-methylcytosine residues using the DYEnamic ET Terminator Kit. A1: untreated pUC19 DNA; A2: M.BamHI N 4 -cytosine methylated pUC19DNA; A3: trace difference between A2 and A1. B1: untreated pUC19DNA; B2: M.HaeIII 5-cytosine methylated pUC19 DNA; B3: trace difference between B2 and B1. The G residues complementary to the methylated cytosines are boxed. N 4 -methylcytosine results in an increase in the complementary G signal, whereas 5-methylcytosine results in a decrease in the complementary G signal.

    Techniques Used: Methylation

    23) Product Images from "Design and Characterization of Bioengineered Cancer-Like Stem Cells"

    Article Title: Design and Characterization of Bioengineered Cancer-Like Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0141172

    Sub-cloning of H ras V12 and LTg into pMSCV plasmids. (A) Genes of interest (i.e. HrasV12 or LTg) were inserted in between MSCV LTRs, and either GFP or RFP gene was used as a tracer gene. (B) Inserts cloned into pMSCV plasmids were confirmed by enzymatic digestions with either BamHI or EcoRI. M: DNA ladder, 1: pMSCV-GFP; 2: pMSCV-H ras V12-GFP; 3: pMSCV-GFP cut ; 4: pMSCV-H ras V12-GFP cut ; 5:pBABE-H ras V12 cut (+ control); 6: pMSCV-RFP; 7: pMSCV-SV40 LTg-RFP; 8: pMSCV-RFP cut 9: pMSCV-SV40 LTg-RFP cut ; 10: pBABE-SV40 LTg cut (+ control). White arrows indicate inserts. Sequences of insert were also verified by DNA sequencing.
    Figure Legend Snippet: Sub-cloning of H ras V12 and LTg into pMSCV plasmids. (A) Genes of interest (i.e. HrasV12 or LTg) were inserted in between MSCV LTRs, and either GFP or RFP gene was used as a tracer gene. (B) Inserts cloned into pMSCV plasmids were confirmed by enzymatic digestions with either BamHI or EcoRI. M: DNA ladder, 1: pMSCV-GFP; 2: pMSCV-H ras V12-GFP; 3: pMSCV-GFP cut ; 4: pMSCV-H ras V12-GFP cut ; 5:pBABE-H ras V12 cut (+ control); 6: pMSCV-RFP; 7: pMSCV-SV40 LTg-RFP; 8: pMSCV-RFP cut 9: pMSCV-SV40 LTg-RFP cut ; 10: pBABE-SV40 LTg cut (+ control). White arrows indicate inserts. Sequences of insert were also verified by DNA sequencing.

    Techniques Used: Subcloning, Clone Assay, DNA Sequencing

    Sub-cloning of H ras V12 and LTg into pMSCV plasmids. (A) Genes of interest (i.e. HrasV12 or LTg) were inserted in between MSCV LTRs, and either GFP or RFP gene was used as a tracer gene. (B) Inserts cloned into pMSCV plasmids were confirmed by enzymatic digestions with either BamHI or EcoRI. M: DNA ladder, 1: pMSCV-GFP; 2: pMSCV-H ras V12-GFP; 3: pMSCV-GFP cut ; 4: pMSCV-H ras V12-GFP cut ; 5:pBABE-H ras V12 cut (+ control); 6: pMSCV-RFP; 7: pMSCV-SV40 LTg-RFP; 8: pMSCV-RFP cut 9: pMSCV-SV40 LTg-RFP cut ; 10: pBABE-SV40 LTg cut (+ control). White arrows indicate inserts. Sequences of insert were also verified by DNA sequencing.
    Figure Legend Snippet: Sub-cloning of H ras V12 and LTg into pMSCV plasmids. (A) Genes of interest (i.e. HrasV12 or LTg) were inserted in between MSCV LTRs, and either GFP or RFP gene was used as a tracer gene. (B) Inserts cloned into pMSCV plasmids were confirmed by enzymatic digestions with either BamHI or EcoRI. M: DNA ladder, 1: pMSCV-GFP; 2: pMSCV-H ras V12-GFP; 3: pMSCV-GFP cut ; 4: pMSCV-H ras V12-GFP cut ; 5:pBABE-H ras V12 cut (+ control); 6: pMSCV-RFP; 7: pMSCV-SV40 LTg-RFP; 8: pMSCV-RFP cut 9: pMSCV-SV40 LTg-RFP cut ; 10: pBABE-SV40 LTg cut (+ control). White arrows indicate inserts. Sequences of insert were also verified by DNA sequencing.

    Techniques Used: Subcloning, Clone Assay, DNA Sequencing

    24) Product Images from "New Molecular Reporters for Rapid Protein Folding Assays"

    Article Title: New Molecular Reporters for Rapid Protein Folding Assays

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0002387

    GFP insertion constructs. (A) GFP insertion constructs (GFPi) contain Nde I and BamH I cloning sites for accepting test proteins (X) between the C and N-termini of a circular permutant of GFP. Flexible amino acid linkers L1 and L2 GGGSGGGS separate the protein of interest from the two GFP domains, (GFP1 and GFP2). GFPi 9/8 starts at amino-acid position 173 (beginning of beta strand 9) and ends at amino-acid position 172 (end of beta strand 8) of GFP ( top ). GFP insertion 8/7, starts at amino-acid 157 (beginning of strand 8), and ends at amino-acid 156 (end of strand 7) ( bottom ). (B) Chimeric GFP insertion constructs of folding reporter GFP ( light gray ) and superfolder GFP ( dark gray ) scaffoldings (FR = folding reporter GFP, SF = superfolder GFP).
    Figure Legend Snippet: GFP insertion constructs. (A) GFP insertion constructs (GFPi) contain Nde I and BamH I cloning sites for accepting test proteins (X) between the C and N-termini of a circular permutant of GFP. Flexible amino acid linkers L1 and L2 GGGSGGGS separate the protein of interest from the two GFP domains, (GFP1 and GFP2). GFPi 9/8 starts at amino-acid position 173 (beginning of beta strand 9) and ends at amino-acid position 172 (end of beta strand 8) of GFP ( top ). GFP insertion 8/7, starts at amino-acid 157 (beginning of strand 8), and ends at amino-acid 156 (end of strand 7) ( bottom ). (B) Chimeric GFP insertion constructs of folding reporter GFP ( light gray ) and superfolder GFP ( dark gray ) scaffoldings (FR = folding reporter GFP, SF = superfolder GFP).

    Techniques Used: Construct, Clone Assay

    25) Product Images from "Quantitative assessment on the cloning efficiencies of lentiviral transfer vectors with a unique clone site"

    Article Title: Quantitative assessment on the cloning efficiencies of lentiviral transfer vectors with a unique clone site

    Journal: Scientific Reports

    doi: 10.1038/srep00415

    Schematic representation of the LV cloning strategy. There is a BamH I clone site at the 5'-end of the original vectors pcDNA3.1/V5-His-Snk/hPlk2, and pEGFP-N1, respectively. A BamH I clone site was inserted at the 3'-ends of EGFP and hPlk2 WT by SDM, respectively. Then, K111M, T239D and T239V mutants were created through SDM using hPlk2 WT gene as template. The inserts were digested with BamH I, and purified. At the same time, pWPI vector was digested by BamH I, and treated with CIP to protect the self-circularization of the vector DNA. Finally, LVs were cloned through ligation and transformation.
    Figure Legend Snippet: Schematic representation of the LV cloning strategy. There is a BamH I clone site at the 5'-end of the original vectors pcDNA3.1/V5-His-Snk/hPlk2, and pEGFP-N1, respectively. A BamH I clone site was inserted at the 3'-ends of EGFP and hPlk2 WT by SDM, respectively. Then, K111M, T239D and T239V mutants were created through SDM using hPlk2 WT gene as template. The inserts were digested with BamH I, and purified. At the same time, pWPI vector was digested by BamH I, and treated with CIP to protect the self-circularization of the vector DNA. Finally, LVs were cloned through ligation and transformation.

    Techniques Used: Clone Assay, Purification, Plasmid Preparation, Ligation, Transformation Assay

    26) Product Images from "SETD2-dependent H3K36me3 plays a critical role in epigenetic regulation of the HPV31 life cycle"

    Article Title: SETD2-dependent H3K36me3 plays a critical role in epigenetic regulation of the HPV31 life cycle

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007367

    SETD2 is necessary for productive viral replication. (A) CIN612 cells were transiently transduced with either a scramble control shRNA (shScram) or one of two SETD2 shRNAs (shSetd2#1 and shSetd2#2) for 72hrs. At this time, DNA and protein were either harvested as an undifferentiated (T0) sample, or cells were grown in high calcium medium to induce differentiation (72hr). DNA was digested with BamHI, which does not cut the viral genome, and Southern blot analysis was performed using the HPV31 genome as a probe. Lysates harvested at the indicated time points were analyzed by immunoblotting to demonstrate the decrease in SETD2 and H3K36me3 upon shRNA-mediated knockdown. Involucrin and K10 were used as markers of differentiation, and p84 and histone H3.1 (H3.1) served as loading controls. (B) CIN612 cells were transduced with lentivirus expressing either control guide RNAs (sgCTR) or guide RNAs targeting SETD2 (sgSETD2 #1 and sgSETD2 #2) and selected with puromycin. Following selection, DNA and protein were harvested from the heterogenous population of cells. DNA was digested with BamHI (non-cutter) and Southern blot analysis performed using the HPV31 genome as a probe. Western blot analysis was performed to examine the levels of SETD2, involucrin and K10 as differentiation controls, with GAPDH as a loading control. (A, B) Fold change in episome copy number for SETD2 knockdown using shRNAs as well as guide RNAs was determined by performing densitometry of episomal bands from three independent experiments using ImageJ software. Shown is the fold change relative to shScram T0 (A) and sgCTR T0 (B), which are set to one. Error bars represent means +/- standard error. Statistics were assayed using a student’s t test. *p≤ .05. WB = western blot. Ca = calcium.
    Figure Legend Snippet: SETD2 is necessary for productive viral replication. (A) CIN612 cells were transiently transduced with either a scramble control shRNA (shScram) or one of two SETD2 shRNAs (shSetd2#1 and shSetd2#2) for 72hrs. At this time, DNA and protein were either harvested as an undifferentiated (T0) sample, or cells were grown in high calcium medium to induce differentiation (72hr). DNA was digested with BamHI, which does not cut the viral genome, and Southern blot analysis was performed using the HPV31 genome as a probe. Lysates harvested at the indicated time points were analyzed by immunoblotting to demonstrate the decrease in SETD2 and H3K36me3 upon shRNA-mediated knockdown. Involucrin and K10 were used as markers of differentiation, and p84 and histone H3.1 (H3.1) served as loading controls. (B) CIN612 cells were transduced with lentivirus expressing either control guide RNAs (sgCTR) or guide RNAs targeting SETD2 (sgSETD2 #1 and sgSETD2 #2) and selected with puromycin. Following selection, DNA and protein were harvested from the heterogenous population of cells. DNA was digested with BamHI (non-cutter) and Southern blot analysis performed using the HPV31 genome as a probe. Western blot analysis was performed to examine the levels of SETD2, involucrin and K10 as differentiation controls, with GAPDH as a loading control. (A, B) Fold change in episome copy number for SETD2 knockdown using shRNAs as well as guide RNAs was determined by performing densitometry of episomal bands from three independent experiments using ImageJ software. Shown is the fold change relative to shScram T0 (A) and sgCTR T0 (B), which are set to one. Error bars represent means +/- standard error. Statistics were assayed using a student’s t test. *p≤ .05. WB = western blot. Ca = calcium.

    Techniques Used: Transduction, shRNA, Southern Blot, Expressing, Selection, Western Blot, Software

    H3K36me3 is required for productive viral replication. (A) CIN612 cells were left untreated (UT) or transduced with lentivirus expressing either wild-type (WT) H3.3 or the H3.3K36M mutant. 72hr post-transduction, cells were harvested as a T0 (undifferentiated) or differentiated in high calcium medium for 72hr. (B) CIN612 cells were transduced with either pLenti-control (CTR) or pLenti-FLAG-KDM4A. Following selection in puromycin, cells were harvested as a T0 (undifferentiated) or were induced in high calcium for 72hr. For (A) and (B) DNA and protein were harvested at the indicated time points. DNA was digested with BamHI (non-cutter) and Southern blotting analysis was performed to analyze episome copy number using the HPV31 genome as a probe. Western blot analysis was performed to examine the levels of H3K36me3, with H3.1 serving as a loading control. Involucrin and K10 were used as markers of differentiation and GAPDH as loading control. For (B) western blot analysis was performed using an antibody to FLAG to detect KDM4A. For (A) and (B), fold change in episome copy number was determined by performing densitometry of episomal bands from three independent experiments using ImageJ software. Graphed is the average fold change relative to (A) UT T0 and (B) pLenti-CTR, which are set to one. Error bars represent means ± standard errors. Statistics were assayed using a student’s t test. * p
    Figure Legend Snippet: H3K36me3 is required for productive viral replication. (A) CIN612 cells were left untreated (UT) or transduced with lentivirus expressing either wild-type (WT) H3.3 or the H3.3K36M mutant. 72hr post-transduction, cells were harvested as a T0 (undifferentiated) or differentiated in high calcium medium for 72hr. (B) CIN612 cells were transduced with either pLenti-control (CTR) or pLenti-FLAG-KDM4A. Following selection in puromycin, cells were harvested as a T0 (undifferentiated) or were induced in high calcium for 72hr. For (A) and (B) DNA and protein were harvested at the indicated time points. DNA was digested with BamHI (non-cutter) and Southern blotting analysis was performed to analyze episome copy number using the HPV31 genome as a probe. Western blot analysis was performed to examine the levels of H3K36me3, with H3.1 serving as a loading control. Involucrin and K10 were used as markers of differentiation and GAPDH as loading control. For (B) western blot analysis was performed using an antibody to FLAG to detect KDM4A. For (A) and (B), fold change in episome copy number was determined by performing densitometry of episomal bands from three independent experiments using ImageJ software. Graphed is the average fold change relative to (A) UT T0 and (B) pLenti-CTR, which are set to one. Error bars represent means ± standard errors. Statistics were assayed using a student’s t test. * p

    Techniques Used: Transduction, Expressing, Mutagenesis, Selection, Southern Blot, Western Blot, Software

    27) Product Images from "An Inducible Alpha-Synuclein Expressing Neuronal Cell Line Model for Parkinson’s Disease"

    Article Title: An Inducible Alpha-Synuclein Expressing Neuronal Cell Line Model for Parkinson’s Disease

    Journal: Journal of Alzheimer's disease : JAD

    doi: 10.3233/JAD-180610

    Generation and characterization of inducible α-synuclein (α-Syn) expressing neuronal line. A) Schematic representation of doxycycline-inducible pCW-iFLAG-α-Syn vector used to generate SHSY-5Y stable cell line and immunoblot showing time-dependent induction of FLAG α-synuclein. B) Densitometry analysis of FLAG and endogenous α-synuclein immunocontent normalized to GAPDH. C) Immunofluorescence of oligomer conformations upon α-synuclein expression. Anti-oligomer A11 antibody recognizes all types of oligomers, but not monomers and fibrils in the case of α-synuclein. D) Oligo A11 antibody fluorescence intensity per cells. Measurement from 30 cells from three different fields.
    Figure Legend Snippet: Generation and characterization of inducible α-synuclein (α-Syn) expressing neuronal line. A) Schematic representation of doxycycline-inducible pCW-iFLAG-α-Syn vector used to generate SHSY-5Y stable cell line and immunoblot showing time-dependent induction of FLAG α-synuclein. B) Densitometry analysis of FLAG and endogenous α-synuclein immunocontent normalized to GAPDH. C) Immunofluorescence of oligomer conformations upon α-synuclein expression. Anti-oligomer A11 antibody recognizes all types of oligomers, but not monomers and fibrils in the case of α-synuclein. D) Oligo A11 antibody fluorescence intensity per cells. Measurement from 30 cells from three different fields.

    Techniques Used: Expressing, Plasmid Preparation, Stable Transfection, Immunofluorescence, Fluorescence

    28) Product Images from "Impacts of the Staphylococcal Enterotoxin H on the Apoptosis and lncRNAs in PC3 and ACHN"

    Article Title: Impacts of the Staphylococcal Enterotoxin H on the Apoptosis and lncRNAs in PC3 and ACHN

    Journal: Molecular Genetics, Microbiology and Virology

    doi: 10.3103/S0891416820030076

    Confirmation of the recombinant plasmid pcDNA3.1(+)- seh by double restriction endonuclease analysis. Line 1, double restriction endonuclease analysis with BamHI / EcoRV for pcDNA3.1(+)- seh (recombinant vector); Line 2, DNA size marker.
    Figure Legend Snippet: Confirmation of the recombinant plasmid pcDNA3.1(+)- seh by double restriction endonuclease analysis. Line 1, double restriction endonuclease analysis with BamHI / EcoRV for pcDNA3.1(+)- seh (recombinant vector); Line 2, DNA size marker.

    Techniques Used: Recombinant, Plasmid Preparation, Marker

    29) Product Images from "Mechanism of Human Papillomavirus Binding to Human Spermatozoa and Fertilizing Ability of Infected Spermatozoa"

    Article Title: Mechanism of Human Papillomavirus Binding to Human Spermatozoa and Fertilizing Ability of Infected Spermatozoa

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0015036

    Sperm transfected with E6/E7 plasmid penetrate oocytes. a . Scheme of the recombinant plasmid pIRES2-AcGFP1-E6E7. E6/E7 genes have been amplified (1032 bp) from plasmid p1321 HPV-16 E6/E7 by PCR and subcloned to plasmid pIRES2-AcGFP1 between SalI and BamHI restriction sites. b . PCR for HPV E6/E7 genes from transfected sperm. Lane M: DNA marker (100 bp); 1: sperm transfected with recombinant E6/E7 plasmid; 2: negative control (no template); 3: sperm transfected only with Lipofectamine 2000; 4: positive control (pIRES2-AcGFP1-E6E7 plasmid). c . Mean number of human sperm penetrated per hamster oocyte in control and sperm transfected with HPV-16 E6/E7 plasmid. d . Hamster oocytes penetrated by control sperm and sperm transfected with HPV16 E6/E7 plasmid in bright field (BF, upper panel) and fluorescence (FL, lower panel) using SYBR green DNA stain.
    Figure Legend Snippet: Sperm transfected with E6/E7 plasmid penetrate oocytes. a . Scheme of the recombinant plasmid pIRES2-AcGFP1-E6E7. E6/E7 genes have been amplified (1032 bp) from plasmid p1321 HPV-16 E6/E7 by PCR and subcloned to plasmid pIRES2-AcGFP1 between SalI and BamHI restriction sites. b . PCR for HPV E6/E7 genes from transfected sperm. Lane M: DNA marker (100 bp); 1: sperm transfected with recombinant E6/E7 plasmid; 2: negative control (no template); 3: sperm transfected only with Lipofectamine 2000; 4: positive control (pIRES2-AcGFP1-E6E7 plasmid). c . Mean number of human sperm penetrated per hamster oocyte in control and sperm transfected with HPV-16 E6/E7 plasmid. d . Hamster oocytes penetrated by control sperm and sperm transfected with HPV16 E6/E7 plasmid in bright field (BF, upper panel) and fluorescence (FL, lower panel) using SYBR green DNA stain.

    Techniques Used: Transfection, Plasmid Preparation, Recombinant, Amplification, Polymerase Chain Reaction, Marker, Negative Control, Positive Control, Fluorescence, SYBR Green Assay, Staining

    30) Product Images from "Determining DNA Supercoiling Enthalpy by Isothermal Titration Calorimetry"

    Article Title: Determining DNA Supercoiling Enthalpy by Isothermal Titration Calorimetry

    Journal: Biochimie

    doi: 10.1016/j.biochi.2012.08.002

    (A) The plasmid map of pXXZ6. Restriction enzyme recognition sites of BamHI, HindIII, and Nt.BbvCI are shown. For supercoiled pXXZ6 used in this study, the supercoiling density was determined to be −0.061. (B) DNA intercalator ethidium bromide
    Figure Legend Snippet: (A) The plasmid map of pXXZ6. Restriction enzyme recognition sites of BamHI, HindIII, and Nt.BbvCI are shown. For supercoiled pXXZ6 used in this study, the supercoiling density was determined to be −0.061. (B) DNA intercalator ethidium bromide

    Techniques Used: Plasmid Preparation

    31) Product Images from "Quantitative assessment on the cloning efficiencies of lentiviral transfer vectors with a unique clone site"

    Article Title: Quantitative assessment on the cloning efficiencies of lentiviral transfer vectors with a unique clone site

    Journal: Scientific Reports

    doi: 10.1038/srep00415

    Schematic representation of the LV cloning strategy. There is a BamH I clone site at the 5'-end of the original vectors pcDNA3.1/V5-His-Snk/hPlk2, and pEGFP-N1, respectively. A BamH I clone site was inserted at the 3'-ends of EGFP and hPlk2 WT by SDM, respectively. Then, K111M, T239D and T239V mutants were created through SDM using hPlk2 WT gene as template. The inserts were digested with BamH I, and purified. At the same time, pWPI vector was digested by BamH I, and treated with CIP to protect the self-circularization of the vector DNA. Finally, LVs were cloned through ligation and transformation.
    Figure Legend Snippet: Schematic representation of the LV cloning strategy. There is a BamH I clone site at the 5'-end of the original vectors pcDNA3.1/V5-His-Snk/hPlk2, and pEGFP-N1, respectively. A BamH I clone site was inserted at the 3'-ends of EGFP and hPlk2 WT by SDM, respectively. Then, K111M, T239D and T239V mutants were created through SDM using hPlk2 WT gene as template. The inserts were digested with BamH I, and purified. At the same time, pWPI vector was digested by BamH I, and treated with CIP to protect the self-circularization of the vector DNA. Finally, LVs were cloned through ligation and transformation.

    Techniques Used: Clone Assay, Purification, Plasmid Preparation, Ligation, Transformation Assay

    32) Product Images from "C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents"

    Article Title: C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1097

    Experimental design for the Bioanalyzer 2100 to identify site-specific endonuclease inhibition by opioid compounds, HindIII, EcoRI, BamHI and SalI.
    Figure Legend Snippet: Experimental design for the Bioanalyzer 2100 to identify site-specific endonuclease inhibition by opioid compounds, HindIII, EcoRI, BamHI and SalI.

    Techniques Used: Inhibition

    ( A ) Electrograms generated using the Bioanalyzer 2100 of 742 bp dsDNA fragment with treatment by endonucleases BamHI, HindIII, SalI and EcoRI. Electrograms of the 742 bp fragment were pre-incubated for 5 h with either ( B ) MC3 , ( C ) HC3 and ( D ) OC3 , followed by exposure over night to the type II restriction endonuclease.
    Figure Legend Snippet: ( A ) Electrograms generated using the Bioanalyzer 2100 of 742 bp dsDNA fragment with treatment by endonucleases BamHI, HindIII, SalI and EcoRI. Electrograms of the 742 bp fragment were pre-incubated for 5 h with either ( B ) MC3 , ( C ) HC3 and ( D ) OC3 , followed by exposure over night to the type II restriction endonuclease.

    Techniques Used: Generated, Incubation

    33) Product Images from "Ruler elements in chromatin remodelers set nucleosome array spacing and phasing"

    Article Title: Ruler elements in chromatin remodelers set nucleosome array spacing and phasing

    Journal: bioRxiv

    doi: 10.1101/2020.02.28.969618

    Quantification of barrier-aligned nucleosome array features depending on barrier, remodeler and nucleosome density. (A) Composite plots of same MNase-seq data for INO80 as in Figure 1D but aligned at anti-Reb1 SLIM-ChlP-defined Reb1 sites (left), or at BamHI sites (right) of SGD chromatin reconstituted at the indicated nucleosome densities and incubated with INO80 and BamHI. (B) Scheme defining array features quantified from barrier-aligned composite plots as in panel A. (C) – (D) Array feature values for the indicated combinations of barrier, remodeler and nucleosome density plotted in different ways allowing comparison between barriers (especially panel C), values (especially panel D) and remodelers (especially panel E). Chd1 refers to the Chd1/FACT complex. Panel D and Figure S2A-C show individual replicates, panels C and E replicate averages.
    Figure Legend Snippet: Quantification of barrier-aligned nucleosome array features depending on barrier, remodeler and nucleosome density. (A) Composite plots of same MNase-seq data for INO80 as in Figure 1D but aligned at anti-Reb1 SLIM-ChlP-defined Reb1 sites (left), or at BamHI sites (right) of SGD chromatin reconstituted at the indicated nucleosome densities and incubated with INO80 and BamHI. (B) Scheme defining array features quantified from barrier-aligned composite plots as in panel A. (C) – (D) Array feature values for the indicated combinations of barrier, remodeler and nucleosome density plotted in different ways allowing comparison between barriers (especially panel C), values (especially panel D) and remodelers (especially panel E). Chd1 refers to the Chd1/FACT complex. Panel D and Figure S2A-C show individual replicates, panels C and E replicate averages.

    Techniques Used: Incubation

    associated with Figures 1 and 2. (A) SDS-PAGE analyses of purified remodeler complexes. (B) Composite plots as in Figure 1D for individual replicates and the indicated combinations of remodeler, Reb1 and nucleosome density. “no remodeler” denotes absence of remodeler. (C) Composite plots aligned at in vivo +1 nucleosome positions (left), Reb1 (middle) or BamHI (right) sites for MNase-seq analysis of SGD chromatin assembled at high nucleosome density and incubated with the indicated remodelers as in Figure 2A (no refill) or with doubling remodeler concentration for the second half of incubation time (refill).
    Figure Legend Snippet: associated with Figures 1 and 2. (A) SDS-PAGE analyses of purified remodeler complexes. (B) Composite plots as in Figure 1D for individual replicates and the indicated combinations of remodeler, Reb1 and nucleosome density. “no remodeler” denotes absence of remodeler. (C) Composite plots aligned at in vivo +1 nucleosome positions (left), Reb1 (middle) or BamHI (right) sites for MNase-seq analysis of SGD chromatin assembled at high nucleosome density and incubated with the indicated remodelers as in Figure 2A (no refill) or with doubling remodeler concentration for the second half of incubation time (refill).

    Techniques Used: SDS Page, Purification, In Vivo, Incubation, Concentration Assay

    34) Product Images from "Evidence Suggesting Absence of Mitochondrial DNA Methylation"

    Article Title: Evidence Suggesting Absence of Mitochondrial DNA Methylation

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2017.00166

    BamHI digestion prior to bisulfite sequencing decreases cytosine unconvertion rate. Targeted bisulfite sequencing was used to compare undigested and digested DNA methylation levels at five different regions of the mtDNA from human muscle cells and SKOV3 cells ( N = 3). (A) Drawing displays the mtDNA regions investigated by targeted bisulfite sequencing. (B) Percentage methylation for undigested and digested mtDNA. Full circle represents cytosines in CpG context whereas open circle is cytosines in non-CpG context. Results are presented with a min-max interval and a sign test was used to test for significant methylation differences. D-loop (6–298) P = 2.02E-41 (Lonza) and P = 1.40E-24 (SKOV3); D-loop (279–458): P = 5.00E-12 (Lonza) and P = 8.20E-08 (SKOV3); tRNA-F+12S: P = 9.22E-15 (Lonza) and P = 1.29E-9 (SKOV3); 16S: P = 6.50E-05; ND5: P = 1.70E-05 (Lonza) and P = 9.97E-13 (SKOV3); CYTB: P = 2.96E-09 (Lonza), and P = 6.68E-13 (SKOV3). D-loop (6–298) includes origin of replication and tRNA-F+12S includes heavy strand promoter 2. P H1 : heavy-strand promoter 1; P H2 : Heavy-strand promote 2; P L : Light-strand promoter; OH: Origin of replication from heavy-strand; O L : Origin of replication from light-strand. ND: not determined.
    Figure Legend Snippet: BamHI digestion prior to bisulfite sequencing decreases cytosine unconvertion rate. Targeted bisulfite sequencing was used to compare undigested and digested DNA methylation levels at five different regions of the mtDNA from human muscle cells and SKOV3 cells ( N = 3). (A) Drawing displays the mtDNA regions investigated by targeted bisulfite sequencing. (B) Percentage methylation for undigested and digested mtDNA. Full circle represents cytosines in CpG context whereas open circle is cytosines in non-CpG context. Results are presented with a min-max interval and a sign test was used to test for significant methylation differences. D-loop (6–298) P = 2.02E-41 (Lonza) and P = 1.40E-24 (SKOV3); D-loop (279–458): P = 5.00E-12 (Lonza) and P = 8.20E-08 (SKOV3); tRNA-F+12S: P = 9.22E-15 (Lonza) and P = 1.29E-9 (SKOV3); 16S: P = 6.50E-05; ND5: P = 1.70E-05 (Lonza) and P = 9.97E-13 (SKOV3); CYTB: P = 2.96E-09 (Lonza), and P = 6.68E-13 (SKOV3). D-loop (6–298) includes origin of replication and tRNA-F+12S includes heavy strand promoter 2. P H1 : heavy-strand promoter 1; P H2 : Heavy-strand promote 2; P L : Light-strand promoter; OH: Origin of replication from heavy-strand; O L : Origin of replication from light-strand. ND: not determined.

    Techniques Used: Methylation Sequencing, DNA Methylation Assay, Methylation

    35) Product Images from "TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression"

    Article Title: TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0186568

    pET-BccI untreated and digested. 1 : DNA ladder. 2 : pET-BccI untreated. 3 : pET-BccI digested with BccI. 4 , 5 , 6 : pET-BccI digested with EcoRI, BamHI and HindIII, respectively.
    Figure Legend Snippet: pET-BccI untreated and digested. 1 : DNA ladder. 2 : pET-BccI untreated. 3 : pET-BccI digested with BccI. 4 , 5 , 6 : pET-BccI digested with EcoRI, BamHI and HindIII, respectively.

    Techniques Used: Positron Emission Tomography

    Screening for E . coli colonies transformed with recombinant pET-BccI. Plasmid preparations from cultures inoculated with transformed colonies were digested with BamHI and subjected to electrophoresis. A ) For BRP recombinants pET-BccI screening, the presence of a 283 bp DNA band indicated there was no recombination (A3, A7, A9), whereas the presence of a 636 bp insert DNA band showed a successful recombination (A2, A4, A5, A6, A8). B ) For CAT recombinants pET-BccI screening, again the presence of a 283 bp DNA band indicated there was no recombination (B2, B5, B6, B8), whereas the presence of a 924 bp insert DNA band showed a successful recombination (B3, B4, B7, B9).
    Figure Legend Snippet: Screening for E . coli colonies transformed with recombinant pET-BccI. Plasmid preparations from cultures inoculated with transformed colonies were digested with BamHI and subjected to electrophoresis. A ) For BRP recombinants pET-BccI screening, the presence of a 283 bp DNA band indicated there was no recombination (A3, A7, A9), whereas the presence of a 636 bp insert DNA band showed a successful recombination (A2, A4, A5, A6, A8). B ) For CAT recombinants pET-BccI screening, again the presence of a 283 bp DNA band indicated there was no recombination (B2, B5, B6, B8), whereas the presence of a 924 bp insert DNA band showed a successful recombination (B3, B4, B7, B9).

    Techniques Used: Transformation Assay, Recombinant, Positron Emission Tomography, Plasmid Preparation, Electrophoresis

    The novel protein-expression vector pET-BccI. The pET-26b (+) derived plasmid has a pBR322 origin of replication, which together with the ROP protein regulates the plasmid copy number per bacterial cell. The kanamycin resistance gene enables positive selection of the transformed E . coli cells in the presence of kanamycin. BamHI, EcoRI and HindIII recognition sites, flanking both sites of the T7 promoter, cloning site and T7 terminator cassette, facilitate the screening of the transformed colonies for the recombinant transformants. The cloning site of pET-BccI, composed of two adjacent reverse BccI recognition sites, provides single 5΄-T and C overhangs after digestion with BccI, which are suitable for the ligation of DNA molecules with complementary edges.
    Figure Legend Snippet: The novel protein-expression vector pET-BccI. The pET-26b (+) derived plasmid has a pBR322 origin of replication, which together with the ROP protein regulates the plasmid copy number per bacterial cell. The kanamycin resistance gene enables positive selection of the transformed E . coli cells in the presence of kanamycin. BamHI, EcoRI and HindIII recognition sites, flanking both sites of the T7 promoter, cloning site and T7 terminator cassette, facilitate the screening of the transformed colonies for the recombinant transformants. The cloning site of pET-BccI, composed of two adjacent reverse BccI recognition sites, provides single 5΄-T and C overhangs after digestion with BccI, which are suitable for the ligation of DNA molecules with complementary edges.

    Techniques Used: Expressing, Plasmid Preparation, Positron Emission Tomography, Derivative Assay, Selection, Transformation Assay, Clone Assay, Recombinant, Ligation

    36) Product Images from "Transgene expression in Penaeus monodon cells: evaluation of recombinant baculoviral vectors with shrimp specific hybrid promoters"

    Article Title: Transgene expression in Penaeus monodon cells: evaluation of recombinant baculoviral vectors with shrimp specific hybrid promoters

    Journal: Cytotechnology

    doi: 10.1007/s10616-015-9872-y

    Agarose gel showing, a Linearized plasmid pFastBac 1™ digested with BamH I; b PCR amplified WSSV immediate early gene (Ie1) product of 502-bp size from infected animal (lanes 1-2); c WSSV Ie1 promoter (502-bp) released from pGEM-T vector after restriction digestion with BamH I (lanes 1-3); d IHHNV P2 promoter (116-bp) released from PGEMT-T vector after restriction digestion with BamH I (lanes 1-3); e GFP gene restriction digested from pEGFP N1 with Sal I and Not I; f colony PCR performed for confirming the alignment of inserted GFP gene in pFastBac 1™ vector between Sal I and Not I sites (lanes 1-5); g Ie1-GFP alignment confirmation using Ie1 forward primer and GFP reverse primer (lanes 1-4); h P2-GFP alignment confirmation using P2 forward primer and GFP reverse primer (lanes 1-4); i PCR confirmation of the recombinant bacmid using M13 forward and GFP reverse primers (lane 1: wild type baculovirus tagged with GFP, lane 2: recombinant bacmid with P2 promoter, lane 3: recombinant bacmid with Ie1 promoter)
    Figure Legend Snippet: Agarose gel showing, a Linearized plasmid pFastBac 1™ digested with BamH I; b PCR amplified WSSV immediate early gene (Ie1) product of 502-bp size from infected animal (lanes 1-2); c WSSV Ie1 promoter (502-bp) released from pGEM-T vector after restriction digestion with BamH I (lanes 1-3); d IHHNV P2 promoter (116-bp) released from PGEMT-T vector after restriction digestion with BamH I (lanes 1-3); e GFP gene restriction digested from pEGFP N1 with Sal I and Not I; f colony PCR performed for confirming the alignment of inserted GFP gene in pFastBac 1™ vector between Sal I and Not I sites (lanes 1-5); g Ie1-GFP alignment confirmation using Ie1 forward primer and GFP reverse primer (lanes 1-4); h P2-GFP alignment confirmation using P2 forward primer and GFP reverse primer (lanes 1-4); i PCR confirmation of the recombinant bacmid using M13 forward and GFP reverse primers (lane 1: wild type baculovirus tagged with GFP, lane 2: recombinant bacmid with P2 promoter, lane 3: recombinant bacmid with Ie1 promoter)

    Techniques Used: Agarose Gel Electrophoresis, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Infection, Recombinant

    37) Product Images from "High-resolution Genetic and Physical Map of the Lgn1 Interval in C57BL/6J Implicates Naip2 or Naip5 in Legionella pneumophila Pathogenesis"

    Article Title: High-resolution Genetic and Physical Map of the Lgn1 Interval in C57BL/6J Implicates Naip2 or Naip5 in Legionella pneumophila Pathogenesis

    Journal: Genome Research

    doi:

    Southern blot analysis of BamHI- and EcoRI-digested BAC DNA identifies six copies of Naip exon 11 and five copies of Naip exon 3. Correlation of the restriction fragments with specific Naip loci was done by comparison of the bands on the BAC blot with predicted fragments from genomic sequence and our previous physical map of the 129 Naip ). Horizontal bars and numbers indicate position and size (kb) of fragments in 1-kb ladder molecular weight marker (GIBCO). ( A ) BamHI-digested DNA probed with Naip exon 11 identifies six Naip exon 11 loci. 129 haplotype genomic sequence predicts the observed 14.3-kb fragment mapping to Naip1 , a 9-kb fragment mapping to Naip2 , an 8.6-kb fragment mapping to Naip5 , and a doublet of 3.5 kb and 3.6 kb mapping to Naip6 and Naip3 . The remaining 2.2-kb band maps to Δ Naip , as observed in the 129 haplotype (data not shown). Asterisk indicates vector junction fragments mapping to Naip1 and Naip2. ( B ) EcoRI-digested DN A probed with Naip exon 3 identifies five Naip exon 3 loci. 129 haplotype genomic sequence predicts the observed 10.2-kb fragment mapping to Naip5 , an 8.5-kb fragment mapping to Naip2 , a 7.5-kb fragment mapping to Naip1 , a 7.2-kb fragment mapping to Naip6 , and 2.1-kb mapping to Naip3 .
    Figure Legend Snippet: Southern blot analysis of BamHI- and EcoRI-digested BAC DNA identifies six copies of Naip exon 11 and five copies of Naip exon 3. Correlation of the restriction fragments with specific Naip loci was done by comparison of the bands on the BAC blot with predicted fragments from genomic sequence and our previous physical map of the 129 Naip ). Horizontal bars and numbers indicate position and size (kb) of fragments in 1-kb ladder molecular weight marker (GIBCO). ( A ) BamHI-digested DNA probed with Naip exon 11 identifies six Naip exon 11 loci. 129 haplotype genomic sequence predicts the observed 14.3-kb fragment mapping to Naip1 , a 9-kb fragment mapping to Naip2 , an 8.6-kb fragment mapping to Naip5 , and a doublet of 3.5 kb and 3.6 kb mapping to Naip6 and Naip3 . The remaining 2.2-kb band maps to Δ Naip , as observed in the 129 haplotype (data not shown). Asterisk indicates vector junction fragments mapping to Naip1 and Naip2. ( B ) EcoRI-digested DN A probed with Naip exon 3 identifies five Naip exon 3 loci. 129 haplotype genomic sequence predicts the observed 10.2-kb fragment mapping to Naip5 , an 8.5-kb fragment mapping to Naip2 , a 7.5-kb fragment mapping to Naip1 , a 7.2-kb fragment mapping to Naip6 , and 2.1-kb mapping to Naip3 .

    Techniques Used: Southern Blot, BAC Assay, Sequencing, Molecular Weight, Marker, Plasmid Preparation

    38) Product Images from "High-resolution Genetic and Physical Map of the Lgn1 Interval in C57BL/6J Implicates Naip2 or Naip5 in Legionella pneumophila Pathogenesis"

    Article Title: High-resolution Genetic and Physical Map of the Lgn1 Interval in C57BL/6J Implicates Naip2 or Naip5 in Legionella pneumophila Pathogenesis

    Journal: Genome Research

    doi:

    Southern blot analysis of BamHI- and EcoRI-digested BAC DNA identifies six copies of Naip exon 11 and five copies of Naip exon 3. Correlation of the restriction fragments with specific Naip loci was done by comparison of the bands on the BAC blot with predicted fragments from genomic sequence and our previous physical map of the 129 Naip ). Horizontal bars and numbers indicate position and size (kb) of fragments in 1-kb ladder molecular weight marker (GIBCO). ( A ) BamHI-digested DNA probed with Naip exon 11 identifies six Naip exon 11 loci. 129 haplotype genomic sequence predicts the observed 14.3-kb fragment mapping to Naip1 , a 9-kb fragment mapping to Naip2 , an 8.6-kb fragment mapping to Naip5 , and a doublet of 3.5 kb and 3.6 kb mapping to Naip6 and Naip3 . The remaining 2.2-kb band maps to Δ Naip , as observed in the 129 haplotype (data not shown). Asterisk indicates vector junction fragments mapping to Naip1 and Naip2. ( B ) EcoRI-digested DN A probed with Naip exon 3 identifies five Naip exon 3 loci. 129 haplotype genomic sequence predicts the observed 10.2-kb fragment mapping to Naip5 , an 8.5-kb fragment mapping to Naip2 , a 7.5-kb fragment mapping to Naip1 , a 7.2-kb fragment mapping to Naip6 , and 2.1-kb mapping to Naip3 .
    Figure Legend Snippet: Southern blot analysis of BamHI- and EcoRI-digested BAC DNA identifies six copies of Naip exon 11 and five copies of Naip exon 3. Correlation of the restriction fragments with specific Naip loci was done by comparison of the bands on the BAC blot with predicted fragments from genomic sequence and our previous physical map of the 129 Naip ). Horizontal bars and numbers indicate position and size (kb) of fragments in 1-kb ladder molecular weight marker (GIBCO). ( A ) BamHI-digested DNA probed with Naip exon 11 identifies six Naip exon 11 loci. 129 haplotype genomic sequence predicts the observed 14.3-kb fragment mapping to Naip1 , a 9-kb fragment mapping to Naip2 , an 8.6-kb fragment mapping to Naip5 , and a doublet of 3.5 kb and 3.6 kb mapping to Naip6 and Naip3 . The remaining 2.2-kb band maps to Δ Naip , as observed in the 129 haplotype (data not shown). Asterisk indicates vector junction fragments mapping to Naip1 and Naip2. ( B ) EcoRI-digested DN A probed with Naip exon 3 identifies five Naip exon 3 loci. 129 haplotype genomic sequence predicts the observed 10.2-kb fragment mapping to Naip5 , an 8.5-kb fragment mapping to Naip2 , a 7.5-kb fragment mapping to Naip1 , a 7.2-kb fragment mapping to Naip6 , and 2.1-kb mapping to Naip3 .

    Techniques Used: Southern Blot, BAC Assay, Sequencing, Molecular Weight, Marker, Plasmid Preparation

    39) Product Images from "RNA aptamer inhibitors of a restriction endonuclease"

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv702

    In vitro selection process. ( A ) RNA aptamer library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), KpnI (green triangles) and PacI (red squares), as a function of selection round.
    Figure Legend Snippet: In vitro selection process. ( A ) RNA aptamer library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), KpnI (green triangles) and PacI (red squares), as a function of selection round.

    Techniques Used: In Vitro, Selection

    40) Product Images from "Expression of the Conserved Herpesvirus Protein Kinase (CHPK) of Marek’s Disease Alphaherpesvirus in the Skin Reveals a Mechanistic Importance for CHPK during Interindividual Spread in Chickens"

    Article Title: Expression of the Conserved Herpesvirus Protein Kinase (CHPK) of Marek’s Disease Alphaherpesvirus in the Skin Reveals a Mechanistic Importance for CHPK during Interindividual Spread in Chickens

    Journal: Journal of Virology

    doi: 10.1128/JVI.01522-19

    ) fragment incorporates two additional BamHI sites, resulting in the reduction of the C fragment (red arrowhead) from 14,427 bp to 9,171 (green asterisk), 5,388 (green asterisk), and 1,070 bp (not shown). Following resolution, only the 1,070-bp fragment is removed. The molecular weight marker used was the GeneRuler 1 kb Plus DNA Ladder from Thermo Scientific (Carlsbad, CA). No extraneous alterations are evident. Kan, kanamycin.
    Figure Legend Snippet: ) fragment incorporates two additional BamHI sites, resulting in the reduction of the C fragment (red arrowhead) from 14,427 bp to 9,171 (green asterisk), 5,388 (green asterisk), and 1,070 bp (not shown). Following resolution, only the 1,070-bp fragment is removed. The molecular weight marker used was the GeneRuler 1 kb Plus DNA Ladder from Thermo Scientific (Carlsbad, CA). No extraneous alterations are evident. Kan, kanamycin.

    Techniques Used: Molecular Weight, Marker

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Chlamydomonas reinhardtii hydin is a central pair protein required for flagellar motility
    Article Snippet: .. The PCR product was digested with HindIII and BamHI and ligated into pMAL-cR1 v. 2 digested with the same enzymes (New England Biolabs, Inc.). .. The construct was transformed into E. coli XL1 blue, and, for expression of the maltose-binding∷hydin fusion protein, into BL21.

    Clone Assay:

    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein
    Article Snippet: .. Both plasmid PGEX-6P-1 (Novagen) and CP were digested with BamH I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in PGEX-6P-1 (PGEX-6P-1-WT-GST-TMV-CP32 ). ..

    other:

    Article Title: Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins
    Article Snippet: Materials Escherichia coli topoisomerase I (Ec TopoI), T4 polynucleotide kinase (PNK), calf intestinal phosphatase, T4 DNA ligase, DNAse I, BamHI, BglII and HindIII were from New England Biolabs.

    Plasmid Preparation:

    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein
    Article Snippet: .. Both plasmid PGEX-6P-1 (Novagen) and CP were digested with BamH I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in PGEX-6P-1 (PGEX-6P-1-WT-GST-TMV-CP32 ). ..

    Article Title: Recognition of DNA Termini by the C-Terminal Region of the Ku80 and the DNA-Dependent Protein Kinase Catalytic Subunit
    Article Snippet: .. Kinase assays using plasmid DNA substrates were performed with pcDNA3.1 digested with either XhoI, BamHI, EcoRV, and KpnI or pCAG-GFP digested with XbaI or EcoRI (New England Biolabs). .. The specific sequences recognized by the restriction enzymes and DNA termini generated are shown in .

    Article Title: Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae
    Article Snippet: .. The ura3 -deficient S. cerevisiae strain BJ5465 ( α ura3–52 trp1 leu2Δ1 his3Δ200 pep4::HIS2 prb1Δ1.6R can1 GAL1 ) was obtained from LGCPromochem, the NucleoSpin Plasmid kit was purchased from Macherey-Nagel, and the restriction enzymes BamHI, NheI, SpeI, SacI, and NotI from New England Biolabs. ..

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    New England Biolabs bamhi
    Results of ddPCR with undigested and digested genomic DNA. Results of ddPCR with undigested genomic DNA showed one copy per genome of Cre (blue circles and line). One copy per genome of Cre was detected by ddPCR after digestion with <t>HindIII</t> (red circles and line) and after digestion with <t>BamHI</t> (green circle and line). Results of three independent experiments for each digestion condition are shown
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    Results of ddPCR with undigested and digested genomic DNA. Results of ddPCR with undigested genomic DNA showed one copy per genome of Cre (blue circles and line). One copy per genome of Cre was detected by ddPCR after digestion with HindIII (red circles and line) and after digestion with BamHI (green circle and line). Results of three independent experiments for each digestion condition are shown

    Journal: Adipocyte

    Article Title: Characterization of the adiponectin promoter + Cre recombinase insertion in the Tg(Adipoq-cre)1Evdr mouse by targeted locus amplification and droplet digital PCR

    doi: 10.1080/21623945.2020.1861728

    Figure Lengend Snippet: Results of ddPCR with undigested and digested genomic DNA. Results of ddPCR with undigested genomic DNA showed one copy per genome of Cre (blue circles and line). One copy per genome of Cre was detected by ddPCR after digestion with HindIII (red circles and line) and after digestion with BamHI (green circle and line). Results of three independent experiments for each digestion condition are shown

    Article Snippet: To separate possible conjoined Cre sequences, the DNA was digested with DNA restriction enzymes with known sites in the targeting BAC sequence both 5ʹ and 3ʹ to the Cre sequence: HindIII (NEB, Ipswich, MA, catalogue #R0104S) in one experiment and BamHI (NEB, Ipswich, MA, catalogue R0136S) in a second experiment.

    Techniques:

    Genomic DNA of Pectobacterium phages CB1, CB3, and CB4, BamHI-digested (lanes 3, 5, and 7, respectively) and undigested (lanes 2, 4, and 6, respectively). Lane 1, DNA marker (Hyperladder 1 kb, Bioline). Gel concentration 1% w / v agarose.

    Journal: Pharmaceuticals

    Article Title: Novel N4-Like Bacteriophages of Pectobacterium atrosepticum

    doi: 10.3390/ph11020045

    Figure Lengend Snippet: Genomic DNA of Pectobacterium phages CB1, CB3, and CB4, BamHI-digested (lanes 3, 5, and 7, respectively) and undigested (lanes 2, 4, and 6, respectively). Lane 1, DNA marker (Hyperladder 1 kb, Bioline). Gel concentration 1% w / v agarose.

    Article Snippet: DNA samples were digested with BamHI and ClaI, according to manufacturer’s protocols (New England BioLabs, Ipswich, MA, USA).

    Techniques: Marker, Concentration Assay

    Sites of structural changes induced by the hyper-negative supercoiling detected by Nuclease SI. (A) Experimental scheme. The red-filled circle designates 32 P. The different steps of the experiment are indicated: first (1), the digestion by the Nuclease SI; second (2), the digestion by (BamHI + BglII) or (BahmHI + HindIII); third (3), electrophoresis on a sequencing gel. (B) The enzymatic probe used to map the fine structure of the T -2 and T -6 topoisomers is Nuclease SI. Nuclease SI is at 2 mU microL -1 and DNA at 0.5 nM. After the Nuclease SI reaction, the samples are treated to remove the proteins. The DNAs are precipitated and submitted to the BamHI+HindIII double digestion to only visualize DNA fragments from one of the two radiolabeled strands. The reaction products are analyzed on two different sequencing gels (8% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 5) or the guanines and adenines (G+A lanes; lanes 2 and 6). (C) Same as 3B except that the samples are submitted to the BglII+BamHI double digestion to only visualize DNA fragments from the complementary radiolabeled strands. The reaction products are analyzed on two different sequencing gels (7% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 7) or the guanines and adenines (G+A lanes; lanes 2 and 6).

    Journal: PLoS ONE

    Article Title: Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins

    doi: 10.1371/journal.pone.0202138

    Figure Lengend Snippet: Sites of structural changes induced by the hyper-negative supercoiling detected by Nuclease SI. (A) Experimental scheme. The red-filled circle designates 32 P. The different steps of the experiment are indicated: first (1), the digestion by the Nuclease SI; second (2), the digestion by (BamHI + BglII) or (BahmHI + HindIII); third (3), electrophoresis on a sequencing gel. (B) The enzymatic probe used to map the fine structure of the T -2 and T -6 topoisomers is Nuclease SI. Nuclease SI is at 2 mU microL -1 and DNA at 0.5 nM. After the Nuclease SI reaction, the samples are treated to remove the proteins. The DNAs are precipitated and submitted to the BamHI+HindIII double digestion to only visualize DNA fragments from one of the two radiolabeled strands. The reaction products are analyzed on two different sequencing gels (8% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 5) or the guanines and adenines (G+A lanes; lanes 2 and 6). (C) Same as 3B except that the samples are submitted to the BglII+BamHI double digestion to only visualize DNA fragments from the complementary radiolabeled strands. The reaction products are analyzed on two different sequencing gels (7% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 7) or the guanines and adenines (G+A lanes; lanes 2 and 6).

    Article Snippet: Materials Escherichia coli topoisomerase I (Ec TopoI), T4 polynucleotide kinase (PNK), calf intestinal phosphatase, T4 DNA ligase, DNAse I, BamHI, BglII and HindIII were from New England Biolabs.

    Techniques: Electrophoresis, Sequencing

    Digestion and Southern analyses of the P-SSP7 genome. (A) Schematic genome map showing the positions of the restriction enzyme cleavage sites (red) and the expected fragment sizes after digestion with BamHI alone (top) and both BamHI and PmeI (bottom) based on the revised genome arrangement shown in Fig. 1C. (B) Restriction digestion of the P-SSP7 genome extracted from phage particles (lanes 3 and 4) and the genome cloned into a fosmid (lanes 5 and 6), with BamHI alone (lanes 3 and 5) or with BamHI and PmeI (lanes 4 and 6), separated by pulse field gel electrophoresis. Note that the only difference for digestion of the cloned genome is the presence of an additional fragment corresponding to the size of the fosmid vector. Fragments corresponding to the expected sizes shown in (A) are marked with the appropriate letter designations (a to f). Fragment size markers (M): 1 kb DNA ladder (lane 1) and Lambda DNA cut with HindIII (lane 2), are shown. (C) Southern analyses of the restriction digested DNA in (B) using 4 probes (denoted above the lanes) show that the repeat region appears twice on the genome on the same fragments as the first and last ORFs. The positions of the gene probes on the genome are shown as light blue boxes and the repeat region probe as green boxes in the top panel of (A). Lane numbering and fragment designations are the same as in (B).

    Journal: PLoS ONE

    Article Title: The P-SSP7 Cyanophage Has a Linear Genome with Direct Terminal Repeats

    doi: 10.1371/journal.pone.0036710

    Figure Lengend Snippet: Digestion and Southern analyses of the P-SSP7 genome. (A) Schematic genome map showing the positions of the restriction enzyme cleavage sites (red) and the expected fragment sizes after digestion with BamHI alone (top) and both BamHI and PmeI (bottom) based on the revised genome arrangement shown in Fig. 1C. (B) Restriction digestion of the P-SSP7 genome extracted from phage particles (lanes 3 and 4) and the genome cloned into a fosmid (lanes 5 and 6), with BamHI alone (lanes 3 and 5) or with BamHI and PmeI (lanes 4 and 6), separated by pulse field gel electrophoresis. Note that the only difference for digestion of the cloned genome is the presence of an additional fragment corresponding to the size of the fosmid vector. Fragments corresponding to the expected sizes shown in (A) are marked with the appropriate letter designations (a to f). Fragment size markers (M): 1 kb DNA ladder (lane 1) and Lambda DNA cut with HindIII (lane 2), are shown. (C) Southern analyses of the restriction digested DNA in (B) using 4 probes (denoted above the lanes) show that the repeat region appears twice on the genome on the same fragments as the first and last ORFs. The positions of the gene probes on the genome are shown as light blue boxes and the repeat region probe as green boxes in the top panel of (A). Lane numbering and fragment designations are the same as in (B).

    Article Snippet: The DNA (0.5 µg per reaction) was digested with BamHI and with a combination of BamHI and PmeI (New England Biolabs).

    Techniques: Clone Assay, Nucleic Acid Electrophoresis, Plasmid Preparation, Lambda DNA Preparation

    Schematic illustration of the arrangement of the P-SSP7 genome. (A) Sequencing of the ends of the P-SSP7 genome extracted directly from phage particles. Arrows, and numbers under the arrows, indicate the sequences acquired: Blue from the entire genome and green from end fragments produced by digestion of the genome with the BamHI and PmeI restriction enzymes. The positions of the primers used for sequencing are shown in black type at the beginning of the arrows. Genome numbering for the primers and sequences is that for the originally published sequence [5] . The purple line denotes the 728 bp region found to be upstream of ORF1 in this study, but positioned downstream of ORF54 in the originally published sequence. The repeat regions are shown in red at both ends of the genome. (B) Diagram showing the arrangement of the P-SSP7 genome as originally published (GenBank accession numbers: AY939843.1, [5] and GU071093 [16] . (C) Diagram of the revised genome arrangement based on the results from this study (updated GeneBank submission, accession number: AY939843.2).

    Journal: PLoS ONE

    Article Title: The P-SSP7 Cyanophage Has a Linear Genome with Direct Terminal Repeats

    doi: 10.1371/journal.pone.0036710

    Figure Lengend Snippet: Schematic illustration of the arrangement of the P-SSP7 genome. (A) Sequencing of the ends of the P-SSP7 genome extracted directly from phage particles. Arrows, and numbers under the arrows, indicate the sequences acquired: Blue from the entire genome and green from end fragments produced by digestion of the genome with the BamHI and PmeI restriction enzymes. The positions of the primers used for sequencing are shown in black type at the beginning of the arrows. Genome numbering for the primers and sequences is that for the originally published sequence [5] . The purple line denotes the 728 bp region found to be upstream of ORF1 in this study, but positioned downstream of ORF54 in the originally published sequence. The repeat regions are shown in red at both ends of the genome. (B) Diagram showing the arrangement of the P-SSP7 genome as originally published (GenBank accession numbers: AY939843.1, [5] and GU071093 [16] . (C) Diagram of the revised genome arrangement based on the results from this study (updated GeneBank submission, accession number: AY939843.2).

    Article Snippet: The DNA (0.5 µg per reaction) was digested with BamHI and with a combination of BamHI and PmeI (New England Biolabs).

    Techniques: Sequencing, Produced