xbai  (New England Biolabs)


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    XbaI
    Description:
    XbaI 15 000 units
    Catalog Number:
    r0145l
    Price:
    290
    Size:
    15 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs xbai
    XbaI
    XbaI 15 000 units
    https://www.bioz.com/result/xbai/product/New England Biolabs
    Average 99 stars, based on 664 article reviews
    Price from $9.99 to $1999.99
    xbai - by Bioz Stars, 2020-09
    99/100 stars

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    Images

    1) Product Images from "Construction of a Bioluminescent Labelling Plasmid Vector for Bifidobacteria"

    Article Title: Construction of a Bioluminescent Labelling Plasmid Vector for Bifidobacteria

    Journal: Korean Journal for Food Science of Animal Resources

    doi: 10.5851/kosfa.2018.e17

    Restriction of pTG262 ( luc + ) (A) and physical genetic map of pTG262::pFI2576 rep ( luc + ) (B). M: HyperLadder™ 1 kb (Bioline Reagents Ltd.); 1: pTG262 [CCC (covalently closed circular) type]; 2: pTG262 ( luc + ) (CCC type); 3: pTG262 ( luc + ) (EcoRI-XbaI).
    Figure Legend Snippet: Restriction of pTG262 ( luc + ) (A) and physical genetic map of pTG262::pFI2576 rep ( luc + ) (B). M: HyperLadder™ 1 kb (Bioline Reagents Ltd.); 1: pTG262 [CCC (covalently closed circular) type]; 2: pTG262 ( luc + ) (CCC type); 3: pTG262 ( luc + ) (EcoRI-XbaI).

    Techniques Used: Countercurrent Chromatography

    2) Product Images from "Acinetobacter baumannii Gastrointestinal Colonization Is Facilitated by Secretory IgA Which Is Reductively Dissociated by Bacterial Thioredoxin A"

    Article Title: Acinetobacter baumannii Gastrointestinal Colonization Is Facilitated by Secretory IgA Which Is Reductively Dissociated by Bacterial Thioredoxin A

    Journal: mBio

    doi: 10.1128/mBio.01298-18

    Generation of a trxA deletion mutant. A schematic representation of the WT A. baumannii Ci79 and Δ trxA mutant genome surrounding the trxA locus with predicted fragment sizes detected by probe following either HindIII (white arrowhead) or XbaI (black arrowhead) digestion as well as probe target region (dotted box) (A). Southern blot (B) and Western blot (C) analyses of genomic and protein extracts, respectively, from WT Ci79, ΔtrxA , and ΔtrxA c A. baumannii were subsequently performed. PCR amplification of the trxA gene locus was performed using DNA from WT Ci79, ΔtrxA , and ΔtrxA c A. baumannii isolates. The PCR amplicons were subsequently digested with SalI and subjected to agarose gel electrophoresis (C). Total bacterial proteins were separated by electrophoresis and visualized in a Coomassie blue-stained polyacrylamide gel (D, left panel) or probed with anti-TrxA antibody to detect TrxA expression (D, right panel). Molecular marker sizes for DNA (bp; B and C) and protein (kDa; D) are provided.
    Figure Legend Snippet: Generation of a trxA deletion mutant. A schematic representation of the WT A. baumannii Ci79 and Δ trxA mutant genome surrounding the trxA locus with predicted fragment sizes detected by probe following either HindIII (white arrowhead) or XbaI (black arrowhead) digestion as well as probe target region (dotted box) (A). Southern blot (B) and Western blot (C) analyses of genomic and protein extracts, respectively, from WT Ci79, ΔtrxA , and ΔtrxA c A. baumannii were subsequently performed. PCR amplification of the trxA gene locus was performed using DNA from WT Ci79, ΔtrxA , and ΔtrxA c A. baumannii isolates. The PCR amplicons were subsequently digested with SalI and subjected to agarose gel electrophoresis (C). Total bacterial proteins were separated by electrophoresis and visualized in a Coomassie blue-stained polyacrylamide gel (D, left panel) or probed with anti-TrxA antibody to detect TrxA expression (D, right panel). Molecular marker sizes for DNA (bp; B and C) and protein (kDa; D) are provided.

    Techniques Used: Mutagenesis, Southern Blot, Western Blot, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Electrophoresis, Staining, Expressing, Marker

    3) Product Images from "Generation and Characterization of a Transgenic Pig Carrying a DsRed-Monomer Reporter Gene"

    Article Title: Generation and Characterization of a Transgenic Pig Carrying a DsRed-Monomer Reporter Gene

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0106864

    Transgenic founder pigs carrying the DsRed gene were detected by (a) PCR and (b) Southern blot analysis. The presence of transgenes in DsRed2 pigs were confirmed by using a DsRed primer, which produced a 780-bp PCR product. The genomic DNA of these 2 transgenic pigs (Nos. 1 and 3) were digested using XbaI and DraI. The digested DNA was subsequently hybridized using a 1.1-kb probe.
    Figure Legend Snippet: Transgenic founder pigs carrying the DsRed gene were detected by (a) PCR and (b) Southern blot analysis. The presence of transgenes in DsRed2 pigs were confirmed by using a DsRed primer, which produced a 780-bp PCR product. The genomic DNA of these 2 transgenic pigs (Nos. 1 and 3) were digested using XbaI and DraI. The digested DNA was subsequently hybridized using a 1.1-kb probe.

    Techniques Used: Transgenic Assay, Polymerase Chain Reaction, Southern Blot, Produced

    The DsRed-Monomer transgenic construct pCX-DsRed-Monomer. Arrows indicate the positions of the PCR DsRed primers. XbaI and DraI were restriction enzyme digestion sites. The thick black line indicates the position of the Southern blot probe.
    Figure Legend Snippet: The DsRed-Monomer transgenic construct pCX-DsRed-Monomer. Arrows indicate the positions of the PCR DsRed primers. XbaI and DraI were restriction enzyme digestion sites. The thick black line indicates the position of the Southern blot probe.

    Techniques Used: Transgenic Assay, Construct, Polymerase Chain Reaction, Southern Blot

    4) Product Images from "Characterizing meiotic chromosomes' structure and pairing using a designer sequence optimized for Hi‐C"

    Article Title: Characterizing meiotic chromosomes' structure and pairing using a designer sequence optimized for Hi‐C

    Journal: Molecular Systems Biology

    doi: 10.15252/msb.20188293

    Diagram of the workflow (related to Fig 1 ) Annotation (SK1 background) corresponds to CDS, ARS, telomere regions, retrotransposable elements, mating type loci, tRNA, Sn/Sno RNA, rDNA, ncRNA, intron motives, and TATA boxes. All those features but CDS and transposons were labeled as “forbidden”, preventing any nucleotide substitution in these regions. DpnII, HindIII, SacI, EcoRI, NdeI, SacII, SalI, XbaI, and XhoI. Putative restriction sites are DNA sequences differing with only one base pair from a RS recognized by a RE. The sequence modifications were allowed only in non‐forbidden positions. In CDS, silent mutations were introduced. When two sites overlapped, the minimum changes needed were selected. When possible, we favored A ↔ G and C ↔ T substitutions. A validation step to test whether or not the deletion of one site creates a new site was performed after each modification, and if so, a new modification was sought for. Modifications to generate new sites were also only introduced at non‐forbidden positions. Only silent mutations were introduced within coding regions. 583 × 150 kb windows with 10‐kb overlaps were generated over the entire genome, excluding telomeres and 75 kb from each side of centromeres. Here, 400, 1,500, 2,000 and 6,000 bp. For each 150‐kb window and each interval, the following steps were performed: for each enzyme, for each starting point: putative sites within the first bin of the window (0− 0+spacing). find the putative sites at position n +1 at a distance interval ± 10% from position n until the end of window. For each window, a score is calculated as follows: for each interval, a score is calculated for each enzyme based on the median absolute deviation (MAD). the best enzyme exhibiting the lowest score was chosen for each interval. Each spacing must have a different enzyme, so multiple combinations of enzymes were computed for each window. The window score is calculated as the sum of the four chosen interval scores. A final step of manual curation was performed to introduced PCRTags (Richardson et al , 2017 ).
    Figure Legend Snippet: Diagram of the workflow (related to Fig 1 ) Annotation (SK1 background) corresponds to CDS, ARS, telomere regions, retrotransposable elements, mating type loci, tRNA, Sn/Sno RNA, rDNA, ncRNA, intron motives, and TATA boxes. All those features but CDS and transposons were labeled as “forbidden”, preventing any nucleotide substitution in these regions. DpnII, HindIII, SacI, EcoRI, NdeI, SacII, SalI, XbaI, and XhoI. Putative restriction sites are DNA sequences differing with only one base pair from a RS recognized by a RE. The sequence modifications were allowed only in non‐forbidden positions. In CDS, silent mutations were introduced. When two sites overlapped, the minimum changes needed were selected. When possible, we favored A ↔ G and C ↔ T substitutions. A validation step to test whether or not the deletion of one site creates a new site was performed after each modification, and if so, a new modification was sought for. Modifications to generate new sites were also only introduced at non‐forbidden positions. Only silent mutations were introduced within coding regions. 583 × 150 kb windows with 10‐kb overlaps were generated over the entire genome, excluding telomeres and 75 kb from each side of centromeres. Here, 400, 1,500, 2,000 and 6,000 bp. For each 150‐kb window and each interval, the following steps were performed: for each enzyme, for each starting point: putative sites within the first bin of the window (0− 0+spacing). find the putative sites at position n +1 at a distance interval ± 10% from position n until the end of window. For each window, a score is calculated as follows: for each interval, a score is calculated for each enzyme based on the median absolute deviation (MAD). the best enzyme exhibiting the lowest score was chosen for each interval. Each spacing must have a different enzyme, so multiple combinations of enzymes were computed for each window. The window score is calculated as the sum of the four chosen interval scores. A final step of manual curation was performed to introduced PCRTags (Richardson et al , 2017 ).

    Techniques Used: Labeling, Sequencing, Modification, Generated

    5) Product Images from "Nearest-neighbor amino acids of specificity-determining residues influence the activity of engineered Cre-type recombinases"

    Article Title: Nearest-neighbor amino acids of specificity-determining residues influence the activity of engineered Cre-type recombinases

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-70867-5

    Rationally designed Cre mutants show increased recombination activity in E.coli . ( a ) Schematic drawing of the plasmid assay. Important regions in the plasmids are indicated. Note the reduced size of the plasmid after recombination. The restrictions sites (BsrGI and XbaI) used for cloning indicated Cre-recombinase variants are depicted. The rox target sites are shown as red triangles. CmR, chloramphenicol resistance gene; ori, origin of replication; AraC, arabinose operon regulatory gene. ( b ) Agarose gels of three independently picked clones showing BsrGI and XbaI digested plasmids carrying indicated recombinases. The amount of arabinose added to the growth medium is presented below each band in μg/ml l +-arabinose. The line with two triangles indicates no recombination, whereas the line with one triangle marks the recombined band. Quantifications of the ratios of band intensities (in percent of recombination) are shown to the right for each mutant. The amount of arabinose added to the growth medium is shown on the X-axis. Error bars depict standard deviation from the three independent experiments.
    Figure Legend Snippet: Rationally designed Cre mutants show increased recombination activity in E.coli . ( a ) Schematic drawing of the plasmid assay. Important regions in the plasmids are indicated. Note the reduced size of the plasmid after recombination. The restrictions sites (BsrGI and XbaI) used for cloning indicated Cre-recombinase variants are depicted. The rox target sites are shown as red triangles. CmR, chloramphenicol resistance gene; ori, origin of replication; AraC, arabinose operon regulatory gene. ( b ) Agarose gels of three independently picked clones showing BsrGI and XbaI digested plasmids carrying indicated recombinases. The amount of arabinose added to the growth medium is presented below each band in μg/ml l +-arabinose. The line with two triangles indicates no recombination, whereas the line with one triangle marks the recombined band. Quantifications of the ratios of band intensities (in percent of recombination) are shown to the right for each mutant. The amount of arabinose added to the growth medium is shown on the X-axis. Error bars depict standard deviation from the three independent experiments.

    Techniques Used: Activity Assay, Plasmid Preparation, Clone Assay, Mutagenesis, Standard Deviation

    6) Product Images from "Genetic and phenotypic diversity of Ralstonia solanacearum biovar 2 strains obtained from Dutch waterways"

    Article Title: Genetic and phenotypic diversity of Ralstonia solanacearum biovar 2 strains obtained from Dutch waterways

    Journal: Antonie Van Leeuwenhoek

    doi: 10.1007/s10482-009-9400-1

    a Agarose gel of uncut genomic DNA of R. solancearum strains, showing the two circular replicons. Lane M : H. wingei chromosomal marker, lane 1 : GMI1000 (biovar 3), lane 2 : 1609, lane 3 : 715, lane 4 : KZR-5, lane 5 : PA2, lane 6 : PA5. Run conditions were: 0.8% chromosomal-grade agarose (1× TAE), switch time of 500 s, 3 V/cm, 14°C for 48 h. b Agarose gel showing pulsed field gel electrophoresis profiles of XbaI digested genomic DNA of R. solanacearum strains. Lane M : lambda marker, lane 1 : 715, lane 2 : 1609, lane 3 : KZR-1, lane 4 : KZR-2, lane 5 : KZR-3, lane 6 : KZR-5, lane 7 : PA1, lane 8 : PA2, lane 9 : PA4, lane 10 : PA5, lane 11 : WA19, lane 12 : WC76, lane 13 : WC78. Arrows : polymorphic bands. Run conditions were: 1% pulsed field certified agarose (0.5× TBE), switch time 1–80 s, 6 V/cm, 14°C for 22 h
    Figure Legend Snippet: a Agarose gel of uncut genomic DNA of R. solancearum strains, showing the two circular replicons. Lane M : H. wingei chromosomal marker, lane 1 : GMI1000 (biovar 3), lane 2 : 1609, lane 3 : 715, lane 4 : KZR-5, lane 5 : PA2, lane 6 : PA5. Run conditions were: 0.8% chromosomal-grade agarose (1× TAE), switch time of 500 s, 3 V/cm, 14°C for 48 h. b Agarose gel showing pulsed field gel electrophoresis profiles of XbaI digested genomic DNA of R. solanacearum strains. Lane M : lambda marker, lane 1 : 715, lane 2 : 1609, lane 3 : KZR-1, lane 4 : KZR-2, lane 5 : KZR-3, lane 6 : KZR-5, lane 7 : PA1, lane 8 : PA2, lane 9 : PA4, lane 10 : PA5, lane 11 : WA19, lane 12 : WC76, lane 13 : WC78. Arrows : polymorphic bands. Run conditions were: 1% pulsed field certified agarose (0.5× TBE), switch time 1–80 s, 6 V/cm, 14°C for 22 h

    Techniques Used: Agarose Gel Electrophoresis, Marker, Pulsed-Field Gel, Electrophoresis

    7) Product Images from "Diverse genome structures of Salmonella paratyphi C"

    Article Title: Diverse genome structures of Salmonella paratyphi C

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-8-290

    PFGE separation of cleaved genomic DNA of S. paratyphi C RKS4594 . (A), cleavage with XbaI; (B), cleavage with AvrII.
    Figure Legend Snippet: PFGE separation of cleaved genomic DNA of S. paratyphi C RKS4594 . (A), cleavage with XbaI; (B), cleavage with AvrII.

    Techniques Used:

    8) 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

    Preferential DNA-PK Activation by Terminal Pyrimidines Leads to Increased NHEJ. a) DNA-PK kinase activity is determined with wild type Ku and linearized reporter plasmid digested with restriction enzymes XbaI and EcoRI. DNA termini generated by digestion are depicted below the graph indicating locations of pyrimidines (Py) and purines (Pu). Activity is reported as pmol of phosphate transferred. b) Representative images analyzed for host cell reactivation assay showing GFP and RFP expression. Images were obtained using a 40x objective. Scale bar = 50 μm. c) Quantified results from host cell reactivation assay. Results are reported as ratio of green cells to red fluorescent cells relative to NHEJ activity. Results from XbaI digested DNA are shown in blue and results from EcoRI digested DNA are shown in purple. Data is presented as the mean and SD with asterisks indicating statistically significant differences.
    Figure Legend Snippet: Preferential DNA-PK Activation by Terminal Pyrimidines Leads to Increased NHEJ. a) DNA-PK kinase activity is determined with wild type Ku and linearized reporter plasmid digested with restriction enzymes XbaI and EcoRI. DNA termini generated by digestion are depicted below the graph indicating locations of pyrimidines (Py) and purines (Pu). Activity is reported as pmol of phosphate transferred. b) Representative images analyzed for host cell reactivation assay showing GFP and RFP expression. Images were obtained using a 40x objective. Scale bar = 50 μm. c) Quantified results from host cell reactivation assay. Results are reported as ratio of green cells to red fluorescent cells relative to NHEJ activity. Results from XbaI digested DNA are shown in blue and results from EcoRI digested DNA are shown in purple. Data is presented as the mean and SD with asterisks indicating statistically significant differences.

    Techniques Used: Activation Assay, Non-Homologous End Joining, Activity Assay, Plasmid Preparation, Generated, Host-Cell Reactivation, Expressing

    9) Product Images from "Defining natural species of bacteria: clear-cut genomic boundaries revealed by a turning point in nucleotide sequence divergence"

    Article Title: Defining natural species of bacteria: clear-cut genomic boundaries revealed by a turning point in nucleotide sequence divergence

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-489

    Genomic DNA PFGE patterns of representative Salmonella strains, cleaved with I-CeuI (a), XbaI (b) and SpeI (c), respectively. Lanes: 1, DNA size Marker; 2-5, S. enteritidis (SE310, SE154, SE301, LK5); 6-14, S. pullorum (RKS5078, CDC1983-67, SARB51, 04–6767, NS387, 00–19557, 02–15951, 03–16062, 98–13777); 15–20, S. gallinarum (287/91, RKS5021, SGSC2293, 91–29327, 90–5289, 92–7995).
    Figure Legend Snippet: Genomic DNA PFGE patterns of representative Salmonella strains, cleaved with I-CeuI (a), XbaI (b) and SpeI (c), respectively. Lanes: 1, DNA size Marker; 2-5, S. enteritidis (SE310, SE154, SE301, LK5); 6-14, S. pullorum (RKS5078, CDC1983-67, SARB51, 04–6767, NS387, 00–19557, 02–15951, 03–16062, 98–13777); 15–20, S. gallinarum (287/91, RKS5021, SGSC2293, 91–29327, 90–5289, 92–7995).

    Techniques Used: Marker

    10) Product Images from "Organization of the cpe Locus in CPE-Positive Clostridium perfringens Type C and D Isolates"

    Article Title: Organization of the cpe Locus in CPE-Positive Clostridium perfringens Type C and D Isolates

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0010932

    RFLP analyses of cpe -positive type A, C, and D isolates and type E isolates carrying silent cpe sequences. DNA from each isolate was digested with XbaI prior to conventional agarose gel electrophoresis and Southern blot hybridization with a cpe -specific probe. The migration of molecular weight markers is shown on the left of the blot.
    Figure Legend Snippet: RFLP analyses of cpe -positive type A, C, and D isolates and type E isolates carrying silent cpe sequences. DNA from each isolate was digested with XbaI prior to conventional agarose gel electrophoresis and Southern blot hybridization with a cpe -specific probe. The migration of molecular weight markers is shown on the left of the blot.

    Techniques Used: Agarose Gel Electrophoresis, Southern Blot, Hybridization, Migration, Molecular Weight

    11) Product Images from "Evaluation of Shigella Species Azithromycin CLSI Epidemiological Cutoff Values and Macrolide Resistance Genes"

    Article Title: Evaluation of Shigella Species Azithromycin CLSI Epidemiological Cutoff Values and Macrolide Resistance Genes

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.01422-18

    (A) PFGE of Shigella flexneri NotI-digested chromosomal DNA along with antibiotic antimicrobial sensitivity testing patterns. In the columns headed “Antibiotics,” a gray box indicates a sensitive profile, and the absence of a gray box (white) indicates a resistant profile. The values on the tree represent the percent similarities between the isolates. (B) PFGE of Shigella flexneri XbaI-digested chromosomal DNA along with antibiotic antimicrobial sensitivity testing patterns. In the columns headed “Antibiotics,” a gray box indicates a sensitive profile, and the absence of a gray box (white) indicates a resistant profile. The values on the tree represent the percent similarities between the isolates.
    Figure Legend Snippet: (A) PFGE of Shigella flexneri NotI-digested chromosomal DNA along with antibiotic antimicrobial sensitivity testing patterns. In the columns headed “Antibiotics,” a gray box indicates a sensitive profile, and the absence of a gray box (white) indicates a resistant profile. The values on the tree represent the percent similarities between the isolates. (B) PFGE of Shigella flexneri XbaI-digested chromosomal DNA along with antibiotic antimicrobial sensitivity testing patterns. In the columns headed “Antibiotics,” a gray box indicates a sensitive profile, and the absence of a gray box (white) indicates a resistant profile. The values on the tree represent the percent similarities between the isolates.

    Techniques Used:

    12) Product Images from "MYC regulates ribosome biogenesis and mitochondrial gene expression programs through interaction with Host Cell Factor-1"

    Article Title: MYC regulates ribosome biogenesis and mitochondrial gene expression programs through interaction with Host Cell Factor-1

    Journal: bioRxiv

    doi: 10.1101/2020.06.22.164764

    Validation of switchable Ramos cell lines to study the MYC–HCF-1 interaction. ( A ) In vitro transcribed/translated T7-tagged HCF-1 VIC was incubated with recombinant FLAG-tagged c-MYC, either WT or mutant (4A or VP16 HBM), and immunoprecipitation (IP) performed using anti-FLAG M2 agarose. Western blot of the input lysate, and the IP eluate, was performed using antibodies against the T7 and FLAG tags. ( B ) The translocated MYC locus from Ramos cells is depicted at top, with chromosome 14 (red) and 8 (blue) elements indicated. Beneath is a representation of the locus modification, in either the unswitched (middle) or switched (bottom) states. This switchable allele contains a wild-type (WT) exon 3, a P2A-linked puromycin cassette, and a SV40 polyadenylation (SV40 PA) signal, all of which are flanked by LoxP sites (black triangles). Downstream of the LoxP-flanked region is an HA-tagged mutant exon 3 (mut-Ex3) and a P2A-linked GFP cassette. Activation of CRE-ER T2 results in excision of WT exon 3 and its replacement with mutant exon 3 which carries sequences encoding either WT or mutant (4A or VP16 HBM) MYC protein. ( C ) Comparison of the structure of the parental (non-modified) MYC allele (top) compared to the switchable MYC allele (bottom). XbaI sites used for digestion of genomic DNA in Southern blot are highlighted, as are the complementary sites for the MYC and GFP probes. The expected products of XbaI digestion for the parental line are 6,784 bp for the MYC probe (which detects both the translocated and non-translocated alleles) and nothing for the GFP probe; for correctly-engineered lines the expected sizes are 6,784 bp and 2,942 bp for the MYC probe, and 6,624 bp for the GFP probe. ( D ) Southern blot using GFP and MYC probes on XbaI-digested gDNA from unswitched parental or switchable cells (WT, 4A, and VP16 HBM), with digested positive and negative control plasmids. ( E ) Switchable cells were treated with or without 20 nM 4-OHT (24 hours), fixed using 1% formaldehyde, and subject to flow cytometry. The GFP profiles of the −4-OHT and +4-OHT cells are shown overlaid onto the same axes, with the approximate percentage of GFP-positive cells for 24 hours +4-OHT shown.
    Figure Legend Snippet: Validation of switchable Ramos cell lines to study the MYC–HCF-1 interaction. ( A ) In vitro transcribed/translated T7-tagged HCF-1 VIC was incubated with recombinant FLAG-tagged c-MYC, either WT or mutant (4A or VP16 HBM), and immunoprecipitation (IP) performed using anti-FLAG M2 agarose. Western blot of the input lysate, and the IP eluate, was performed using antibodies against the T7 and FLAG tags. ( B ) The translocated MYC locus from Ramos cells is depicted at top, with chromosome 14 (red) and 8 (blue) elements indicated. Beneath is a representation of the locus modification, in either the unswitched (middle) or switched (bottom) states. This switchable allele contains a wild-type (WT) exon 3, a P2A-linked puromycin cassette, and a SV40 polyadenylation (SV40 PA) signal, all of which are flanked by LoxP sites (black triangles). Downstream of the LoxP-flanked region is an HA-tagged mutant exon 3 (mut-Ex3) and a P2A-linked GFP cassette. Activation of CRE-ER T2 results in excision of WT exon 3 and its replacement with mutant exon 3 which carries sequences encoding either WT or mutant (4A or VP16 HBM) MYC protein. ( C ) Comparison of the structure of the parental (non-modified) MYC allele (top) compared to the switchable MYC allele (bottom). XbaI sites used for digestion of genomic DNA in Southern blot are highlighted, as are the complementary sites for the MYC and GFP probes. The expected products of XbaI digestion for the parental line are 6,784 bp for the MYC probe (which detects both the translocated and non-translocated alleles) and nothing for the GFP probe; for correctly-engineered lines the expected sizes are 6,784 bp and 2,942 bp for the MYC probe, and 6,624 bp for the GFP probe. ( D ) Southern blot using GFP and MYC probes on XbaI-digested gDNA from unswitched parental or switchable cells (WT, 4A, and VP16 HBM), with digested positive and negative control plasmids. ( E ) Switchable cells were treated with or without 20 nM 4-OHT (24 hours), fixed using 1% formaldehyde, and subject to flow cytometry. The GFP profiles of the −4-OHT and +4-OHT cells are shown overlaid onto the same axes, with the approximate percentage of GFP-positive cells for 24 hours +4-OHT shown.

    Techniques Used: In Vitro, Incubation, Recombinant, Mutagenesis, Immunoprecipitation, Western Blot, Modification, Activation Assay, Southern Blot, Negative Control, Flow Cytometry

    13) Product Images from "Identification and Characterization of Spontaneous Deletions within the Sp11-Sp12 Prophage Region of Escherichia coli O157:H7 Sakai"

    Article Title: Identification and Characterization of Spontaneous Deletions within the Sp11-Sp12 Prophage Region of Escherichia coli O157:H7 Sakai

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.03682-12

    Comparison of PFGE profiles of E. coli O157:H7 Sakai and its derivatives. A total of 4 strains were digested using XbaI in this assay. A Salmonella enterica serotype Braenderup reference standard (H9812), also restricted with XbaI, was used as a size standard (M). The approximate size of each marker band is presented in kilobases. The white arrow highlights the 187-kb band observed only with WT Sakai.
    Figure Legend Snippet: Comparison of PFGE profiles of E. coli O157:H7 Sakai and its derivatives. A total of 4 strains were digested using XbaI in this assay. A Salmonella enterica serotype Braenderup reference standard (H9812), also restricted with XbaI, was used as a size standard (M). The approximate size of each marker band is presented in kilobases. The white arrow highlights the 187-kb band observed only with WT Sakai.

    Techniques Used: Marker

    14) Product Images from "USER(TM) friendly DNA engineering and cloning method by uracil excision"

    Article Title: USER(TM) friendly DNA engineering and cloning method by uracil excision

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm041

    Schematic representation of the pNEB206A cloning vector. ( A ) pNEB206A vector was constructed by ligating a synthetic double-stranded cassette into the PacI and PmeI sites of pNEB193. Within the cassette, XbaI and Nt.BbvCI recognition sequences are underlined; cleavage sites are shown by arrows. ( B ) For USER friendly cloning, pNEB206A is double-digested with XbaI and Nt.BbvCI as described in the Materials and methods section to produce linearized vector flanked by 3′ single-stranded extensions on both ends.
    Figure Legend Snippet: Schematic representation of the pNEB206A cloning vector. ( A ) pNEB206A vector was constructed by ligating a synthetic double-stranded cassette into the PacI and PmeI sites of pNEB193. Within the cassette, XbaI and Nt.BbvCI recognition sequences are underlined; cleavage sites are shown by arrows. ( B ) For USER friendly cloning, pNEB206A is double-digested with XbaI and Nt.BbvCI as described in the Materials and methods section to produce linearized vector flanked by 3′ single-stranded extensions on both ends.

    Techniques Used: Clone Assay, Plasmid Preparation, Construct

    15) Product Images from "Generation of a Novel Nucleic Acid-Based Reporter System To Detect Phenotypic Susceptibility to Antibiotics in Mycobacterium tuberculosis"

    Article Title: Generation of a Novel Nucleic Acid-Based Reporter System To Detect Phenotypic Susceptibility to Antibiotics in Mycobacterium tuberculosis

    Journal: mBio

    doi: 10.1128/mBio.00312-11

    Design and characterization of phSP6-ProPol. (A) The SGM was comprised of 2 sections contained within a XbaI - NotI restriction fragment: the SP6 RNA polymerase gene (SP6 RNA Pol) under transcriptional control of the mycobacteriophage L5 P left promoter (open star) and the consensus SP6 promoter fused to the SML-encoding sequence (filled star). The SP6-SML section is flanked by 2 transcription terminators: the upstream terminator (filled circle) is E. coli rrnBT2 and precludes basal transcription through the SML-encoding sequence by host RNA polymerase; the downstream terminator (open circle) is the SP6 RNA polymerase terminator from the region downstream of the SP6 phage major capsid subunit described by Dobbins et al. ( 22 ). After expression of SP6 RNA polymerase from P left , the SML-encoding sequence downstream of the SP6 promoter was transcribed by SP6 RNA polymerase. SP6-dependent transcription of the SML-encoding sequence constituted generation of the SML. SML RNA could then be amplified and detected using primers that bind the SML. (B) The TM4 genome is depicted by the solid black line at the top of the figure. Expression of phage genes occurs on one strand of the genome, and the direction of transcription is indicated by the dashed arrow above the phage genome. Transgenic functions inserted into TM4 are contained on a NotI fragment, which is indicated and expanded. phAE142 encodes an ampicillin resistance cassette (Amp r ) and an origin of replication (oriE) for maintenance and selection of the phasmid in E. coli . phAE142 also encodes the luciferase open reading frame fused to P left . phSP6-ProPol was derived from phAE142 and replaced the luciferase-encoding XbaI - NotI fragment with the XbaI - NotI SGM. In addition, phSP6-ProPol contained a kanamycin resistance cassette (Km r ) in place of phAE142 Amp r . P left transcription occurred on the strand opposite the endogenous phage functions in both phAE142 and phSP6-ProPol. The binding sites and orientation of oligonucleotide primers Ul53-UpSt-113348 and Ul53-DnSt-112112 (UpSt and DnSt, respectively) used for detection of SML generation and those used to characterize transgene structure in phSP6-ProPol are indicated. (C) Phage eluted from primary (1°) plaques originating with transformation of 2 independent phSP6-ProPol-Kan phasmid DNA clones (#4 and #5), as well as phAE142 phasmid DNA, into mc 2 4502 were added to a PCR with the primers P L LF and TM4-50133.52. Phasmid DNA with and without the addition of MP buffer was included as controls. P L LF and TM4-50133.52 were predicted to generate a 667-bp product using phSP6-ProPol as a template, compared to a 181-bp product when phAE142 was the substrate. Products were separated on a 2% agarose gel and visualized by ethidium bromide staining. Locations of DNA size markers are indicated. (D) Phage eluted from the 1° plaques in panel C were amplified using primers P L LU and P L LD, which were predicted to mediate amplification of 2,854-bp and 1,882-bp products in phSP6-ProPol and phAE142, respectively. Products were then separated on a 1% agarose gel and visualized by ethidium bromide staining. The locations of DNA size markers are indicated.
    Figure Legend Snippet: Design and characterization of phSP6-ProPol. (A) The SGM was comprised of 2 sections contained within a XbaI - NotI restriction fragment: the SP6 RNA polymerase gene (SP6 RNA Pol) under transcriptional control of the mycobacteriophage L5 P left promoter (open star) and the consensus SP6 promoter fused to the SML-encoding sequence (filled star). The SP6-SML section is flanked by 2 transcription terminators: the upstream terminator (filled circle) is E. coli rrnBT2 and precludes basal transcription through the SML-encoding sequence by host RNA polymerase; the downstream terminator (open circle) is the SP6 RNA polymerase terminator from the region downstream of the SP6 phage major capsid subunit described by Dobbins et al. ( 22 ). After expression of SP6 RNA polymerase from P left , the SML-encoding sequence downstream of the SP6 promoter was transcribed by SP6 RNA polymerase. SP6-dependent transcription of the SML-encoding sequence constituted generation of the SML. SML RNA could then be amplified and detected using primers that bind the SML. (B) The TM4 genome is depicted by the solid black line at the top of the figure. Expression of phage genes occurs on one strand of the genome, and the direction of transcription is indicated by the dashed arrow above the phage genome. Transgenic functions inserted into TM4 are contained on a NotI fragment, which is indicated and expanded. phAE142 encodes an ampicillin resistance cassette (Amp r ) and an origin of replication (oriE) for maintenance and selection of the phasmid in E. coli . phAE142 also encodes the luciferase open reading frame fused to P left . phSP6-ProPol was derived from phAE142 and replaced the luciferase-encoding XbaI - NotI fragment with the XbaI - NotI SGM. In addition, phSP6-ProPol contained a kanamycin resistance cassette (Km r ) in place of phAE142 Amp r . P left transcription occurred on the strand opposite the endogenous phage functions in both phAE142 and phSP6-ProPol. The binding sites and orientation of oligonucleotide primers Ul53-UpSt-113348 and Ul53-DnSt-112112 (UpSt and DnSt, respectively) used for detection of SML generation and those used to characterize transgene structure in phSP6-ProPol are indicated. (C) Phage eluted from primary (1°) plaques originating with transformation of 2 independent phSP6-ProPol-Kan phasmid DNA clones (#4 and #5), as well as phAE142 phasmid DNA, into mc 2 4502 were added to a PCR with the primers P L LF and TM4-50133.52. Phasmid DNA with and without the addition of MP buffer was included as controls. P L LF and TM4-50133.52 were predicted to generate a 667-bp product using phSP6-ProPol as a template, compared to a 181-bp product when phAE142 was the substrate. Products were separated on a 2% agarose gel and visualized by ethidium bromide staining. Locations of DNA size markers are indicated. (D) Phage eluted from the 1° plaques in panel C were amplified using primers P L LU and P L LD, which were predicted to mediate amplification of 2,854-bp and 1,882-bp products in phSP6-ProPol and phAE142, respectively. Products were then separated on a 1% agarose gel and visualized by ethidium bromide staining. The locations of DNA size markers are indicated.

    Techniques Used: Sequencing, Expressing, Amplification, Transgenic Assay, Selection, Luciferase, Derivative Assay, Binding Assay, Transformation Assay, Clone Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    16) Product Images from "Organization of the cpe Locus in CPE-Positive Clostridium perfringens Type C and D Isolates"

    Article Title: Organization of the cpe Locus in CPE-Positive Clostridium perfringens Type C and D Isolates

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0010932

    RFLP analyses of cpe -positive type A, C, and D isolates and type E isolates carrying silent cpe sequences. DNA from each isolate was digested with XbaI prior to conventional agarose gel electrophoresis and Southern blot hybridization with a cpe -specific probe. The migration of molecular weight markers is shown on the left of the blot.
    Figure Legend Snippet: RFLP analyses of cpe -positive type A, C, and D isolates and type E isolates carrying silent cpe sequences. DNA from each isolate was digested with XbaI prior to conventional agarose gel electrophoresis and Southern blot hybridization with a cpe -specific probe. The migration of molecular weight markers is shown on the left of the blot.

    Techniques Used: Agarose Gel Electrophoresis, Southern Blot, Hybridization, Migration, Molecular Weight

    17) Product Images from "DNA Topoisomerases Participate in Fragility of the Oncogene RET"

    Article Title: DNA Topoisomerases Participate in Fragility of the Oncogene RET

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0075741

    Location of APH-induced DNA breakpoints within intron 11 of RET detected by LM-PCR. (A) The location of 144 APH-induced DNA breakpoints isolated within intron 11 of RET by LM-PCR were determined by DNA sequencing (arrowheads). DNA breaks identified on the strand shown by the sequence are indicated by black arrowheads, and on the complementary strand by grey arrowheads. Open arrowheads indicate the locations of known patient breakpoints observed in PTC tumors containing RET/PTC rearrangements [ 39 , 47 – 50 ]. The location of BanI and XbaI digestion sites within intron 11 are labeled. RET primer sets (see Table S1 ) are indicated by arrows. Lines with circles are dual biotin-labeled primers followed by two nested primers. The dashed black lines represent primer set 1, dashed grey lines primer set 2, solid black lines primer set 3, and solid grey lines primer set 4. The sequence of intron 11 is displayed along with the flanking exon 10 and 11 sequences, shown in italics. (B) The distribution of APH-induced DNA breakpoints within intron 11 are depicted as a smooth curve fit of the percentage of breakpoints (y axis) located every 50 bp of intron 11 in a 5’ to 3’ direction (x axis).
    Figure Legend Snippet: Location of APH-induced DNA breakpoints within intron 11 of RET detected by LM-PCR. (A) The location of 144 APH-induced DNA breakpoints isolated within intron 11 of RET by LM-PCR were determined by DNA sequencing (arrowheads). DNA breaks identified on the strand shown by the sequence are indicated by black arrowheads, and on the complementary strand by grey arrowheads. Open arrowheads indicate the locations of known patient breakpoints observed in PTC tumors containing RET/PTC rearrangements [ 39 , 47 – 50 ]. The location of BanI and XbaI digestion sites within intron 11 are labeled. RET primer sets (see Table S1 ) are indicated by arrows. Lines with circles are dual biotin-labeled primers followed by two nested primers. The dashed black lines represent primer set 1, dashed grey lines primer set 2, solid black lines primer set 3, and solid grey lines primer set 4. The sequence of intron 11 is displayed along with the flanking exon 10 and 11 sequences, shown in italics. (B) The distribution of APH-induced DNA breakpoints within intron 11 are depicted as a smooth curve fit of the percentage of breakpoints (y axis) located every 50 bp of intron 11 in a 5’ to 3’ direction (x axis).

    Techniques Used: Polymerase Chain Reaction, Isolation, DNA Sequencing, Sequencing, Labeling

    18) Product Images from "Construction of a Bioluminescent Labelling Plasmid Vector for Bifidobacteria"

    Article Title: Construction of a Bioluminescent Labelling Plasmid Vector for Bifidobacteria

    Journal: Korean Journal for Food Science of Animal Resources

    doi: 10.5851/kosfa.2018.e17

    Restriction of pTG262 ( luc + ) (A) and physical genetic map of pTG262::pFI2576 rep ( luc + ) (B). M: HyperLadder™ 1 kb (Bioline Reagents Ltd.); 1: pTG262 [CCC (covalently closed circular) type]; 2: pTG262 ( luc + ) (CCC type); 3: pTG262 ( luc + ) (EcoRI-XbaI).
    Figure Legend Snippet: Restriction of pTG262 ( luc + ) (A) and physical genetic map of pTG262::pFI2576 rep ( luc + ) (B). M: HyperLadder™ 1 kb (Bioline Reagents Ltd.); 1: pTG262 [CCC (covalently closed circular) type]; 2: pTG262 ( luc + ) (CCC type); 3: pTG262 ( luc + ) (EcoRI-XbaI).

    Techniques Used: Countercurrent Chromatography

    19) Product Images from "Generation of an Oncolytic Herpes Simplex Virus 1 Expressing Human MelanA"

    Article Title: Generation of an Oncolytic Herpes Simplex Virus 1 Expressing Human MelanA

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00002

    Generation of HSV-1 d 106S-MelanA. (A) ) using XbaI/NheI-containing primers. (B) Light microscopic images of uninfected E11 cells (left) and cytopathic effects induced in these cells 56 h post HSV-1 d 106S infection (right). (C) EcoRI digestion of HSV-1 d 106S DNA obtained from viral nucleocapsids showing distinct bands as evidence of DNA integrity. (D) Overlay of phase contrast and immunofluorescence microscopy of E11 cells harboring fluorescing (upper part) and non-fluorescing (lower part) viral plaques, representing HSV-1 d 106S and HSV-1 d 106S-MelanA, respectively, after cotransfection of the linearized transfer plasmid pd27B-MelanA and HSV-1 d 106S DNA. Light and immunofluorescence microscopy were taken using the DMI 6000B inverted microscope (20 × magnification).
    Figure Legend Snippet: Generation of HSV-1 d 106S-MelanA. (A) ) using XbaI/NheI-containing primers. (B) Light microscopic images of uninfected E11 cells (left) and cytopathic effects induced in these cells 56 h post HSV-1 d 106S infection (right). (C) EcoRI digestion of HSV-1 d 106S DNA obtained from viral nucleocapsids showing distinct bands as evidence of DNA integrity. (D) Overlay of phase contrast and immunofluorescence microscopy of E11 cells harboring fluorescing (upper part) and non-fluorescing (lower part) viral plaques, representing HSV-1 d 106S and HSV-1 d 106S-MelanA, respectively, after cotransfection of the linearized transfer plasmid pd27B-MelanA and HSV-1 d 106S DNA. Light and immunofluorescence microscopy were taken using the DMI 6000B inverted microscope (20 × magnification).

    Techniques Used: Infection, Immunofluorescence, Microscopy, Cotransfection, Plasmid Preparation, Inverted Microscopy

    20) Product Images from "Use of an EZ-Tn5-Based Random Mutagenesis System to Identify a Novel Toxin Regulatory Locus in Clostridium perfringens Strain 13"

    Article Title: Use of an EZ-Tn5-Based Random Mutagenesis System to Identify a Novel Toxin Regulatory Locus in Clostridium perfringens Strain 13

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006232

    Southern blot analyses of C. perfringens random mutants obtained after electroporation with EZ-Tn 5 transposomes. After selection on BHI plates containing Erm (40 µg/ml), DNA was extracted from strain 13 transformants. Following digestion with EcoRI (A) or XbaI (B), the digested DNA was electrophoresed and blotted to a nylon membrane. DNA on the membranes was then hybridized with a Dig-labeled erm probe, as found in the C. perfringens -modified EZ-Tn5, and blots were developed as described in the Materials and Methods . Size of DNA fragments, in kilobases (kb), is shown at left.
    Figure Legend Snippet: Southern blot analyses of C. perfringens random mutants obtained after electroporation with EZ-Tn 5 transposomes. After selection on BHI plates containing Erm (40 µg/ml), DNA was extracted from strain 13 transformants. Following digestion with EcoRI (A) or XbaI (B), the digested DNA was electrophoresed and blotted to a nylon membrane. DNA on the membranes was then hybridized with a Dig-labeled erm probe, as found in the C. perfringens -modified EZ-Tn5, and blots were developed as described in the Materials and Methods . Size of DNA fragments, in kilobases (kb), is shown at left.

    Techniques Used: Southern Blot, Electroporation, Selection, Labeling, Modification

    21) Product Images from "Conservative site-specific and single-copy transgenesis in human LINE-1 elements"

    Article Title: Conservative site-specific and single-copy transgenesis in human LINE-1 elements

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv1345

    Targeting endogenous att H4X in HT1080 cells. ( A ) Diagram showing the 21 nucleotide sequences comprising the core binding and spacer sequences of various att sites and features of the LINE -1 retrotransposon. Wild type att B and att P sequences aligned to show the respective core binding and spacer sequences. The query sequence was used in a bioinformatics search for targets resembling core att sites. Sequence logo analysis was performed for the 18 bp att H4X sequences in LINE-1 elements in the targeted cell lines including additional three nucleotides at the 5′ end in order to compare with the 21 bp att B sequence. A LINE -1 retrotransposon diagram with the position and orientation of att H4X is shown at the bottom. See text for details. ( B ) Schematic drawing showing pattP4X-PGKssPuro target vector and predicted recombination between att P4X and genomic att H4X. Positions of relevant primers (Puro rev24, Puro rev303, Puro fw318, Puro fw509 and pr21), restriction sites and the probe used for Southern blotting are indicated. ( C ) Screening for att H4X × att P4X recombination events in HT1080 clones. PCR was performed with genomic DNA and primers cs_ att H4X_F1 and Puro rev24 (for att L junction) and cs_ att H4X_R1 and pr21 (for att R junction). PCR amplified products of the expected size (1100 bp; for the left junction) were detected in clones 3, 19 and 21 (top two panels) and (∼375 bp; for the right junction) in clones 11, 19 and 21 (bottom two panels). W, no DNA template control; HT, negative control (genomic DNA from parental cells); Ina, genomic DNA from puromycin resistant clones obtained through co-transfection of pattP4X-PGKssPuro and pCMVssIna , the latter expressed inactive Int as negative control; +, positive control (genomic DNA from a HT1080 clone carrying an att H4X x att P4X integration event); M, 100 bp DNA ladder; 1 to 33, genomic DNA from puromycin resistant HT1080 clones obtained through co-transfection of pattP4X-PGKssPuro and pCMVssInt-C3CNLS . ( D ) Southern blot analysis. Genomic DNA purified from five targeted HT1080 clones, as indicated, and parental HT1080 cell line was subjected to digestion with PstI or XbaI. A PCR-derived digoxigenin-labeled probe complementary to the puromycin resistance gene was used. Lanes: M1, 1 kb DNA ladder; HT, genomic DNA from HT1080; 3, 11, 18*, 19 and 21, genomic DNA from targeted clones; pP4X (10 7 , 10 8 , 10 9 ), copies of linearized pattP4X-PGKssPuro loaded as positive control. The arrows indicate fragments of expected size for clones 3 and 11. *HT 1080 Clone 18 (carrying a single-copy transgene) was obtained from a screen of puromycin-resistant HT1080 clones that resulted from a previous co-transfection of pattP4X-PGKssPuro and pCMVssInt-h/218 . White arrow heads indicate fragments of the expected size and black arrow heads indicate extra or unexpected fragments in the targeted clones.
    Figure Legend Snippet: Targeting endogenous att H4X in HT1080 cells. ( A ) Diagram showing the 21 nucleotide sequences comprising the core binding and spacer sequences of various att sites and features of the LINE -1 retrotransposon. Wild type att B and att P sequences aligned to show the respective core binding and spacer sequences. The query sequence was used in a bioinformatics search for targets resembling core att sites. Sequence logo analysis was performed for the 18 bp att H4X sequences in LINE-1 elements in the targeted cell lines including additional three nucleotides at the 5′ end in order to compare with the 21 bp att B sequence. A LINE -1 retrotransposon diagram with the position and orientation of att H4X is shown at the bottom. See text for details. ( B ) Schematic drawing showing pattP4X-PGKssPuro target vector and predicted recombination between att P4X and genomic att H4X. Positions of relevant primers (Puro rev24, Puro rev303, Puro fw318, Puro fw509 and pr21), restriction sites and the probe used for Southern blotting are indicated. ( C ) Screening for att H4X × att P4X recombination events in HT1080 clones. PCR was performed with genomic DNA and primers cs_ att H4X_F1 and Puro rev24 (for att L junction) and cs_ att H4X_R1 and pr21 (for att R junction). PCR amplified products of the expected size (1100 bp; for the left junction) were detected in clones 3, 19 and 21 (top two panels) and (∼375 bp; for the right junction) in clones 11, 19 and 21 (bottom two panels). W, no DNA template control; HT, negative control (genomic DNA from parental cells); Ina, genomic DNA from puromycin resistant clones obtained through co-transfection of pattP4X-PGKssPuro and pCMVssIna , the latter expressed inactive Int as negative control; +, positive control (genomic DNA from a HT1080 clone carrying an att H4X x att P4X integration event); M, 100 bp DNA ladder; 1 to 33, genomic DNA from puromycin resistant HT1080 clones obtained through co-transfection of pattP4X-PGKssPuro and pCMVssInt-C3CNLS . ( D ) Southern blot analysis. Genomic DNA purified from five targeted HT1080 clones, as indicated, and parental HT1080 cell line was subjected to digestion with PstI or XbaI. A PCR-derived digoxigenin-labeled probe complementary to the puromycin resistance gene was used. Lanes: M1, 1 kb DNA ladder; HT, genomic DNA from HT1080; 3, 11, 18*, 19 and 21, genomic DNA from targeted clones; pP4X (10 7 , 10 8 , 10 9 ), copies of linearized pattP4X-PGKssPuro loaded as positive control. The arrows indicate fragments of expected size for clones 3 and 11. *HT 1080 Clone 18 (carrying a single-copy transgene) was obtained from a screen of puromycin-resistant HT1080 clones that resulted from a previous co-transfection of pattP4X-PGKssPuro and pCMVssInt-h/218 . White arrow heads indicate fragments of the expected size and black arrow heads indicate extra or unexpected fragments in the targeted clones.

    Techniques Used: Binding Assay, Sequencing, Plasmid Preparation, Southern Blot, Clone Assay, Polymerase Chain Reaction, Amplification, Negative Control, Cotransfection, Positive Control, Purification, Derivative Assay, Labeling

    Targeting att H4X in hESCs with pattP4X-PGKssPuro-UTF1-eGFP . ( A ) Schematic diagram of pattP4X-PGKssPuro-UTF1-eGFP targeting vector after integration. Locations of primers (Puro rev24, PGK rev, pr111 and pr22) and the Southern probe are indicated. ( B ) Screening for att H4X x att P4X recombination events in selected hESC clones. Semi-nested PCR with primers cs_ att H4X_F1 and PGK rev (for the left junction) using templates obtained with primary PCR (primers cs_ att H4X_F1 and Puro rev24). PCR products of the expected size (∼900 bp) were detected in hESC clone E3 (left panel). Confirmatory PCR with genomic locus specific primers were performed for clone E3. PCR products of expected size (∼1100 bp) were obtained in a semi-nested PCR with primer PGK rev and genomic locus-specific forward primer E3-GF2 using templates from a primary PCR (primers Puro rev24 and primer E3-GF2; middle panel). PCR products of expected size (∼1000 bp) were obtained in a semi-nested PCR with primer pr111 and genomic locus specific reverse primer E3-GR2 (for the right junction) using templates from a primary PCR (primers pr22 and E3-GR2; right panel). W, no DNA template control; ES, negative control (genomic DNA from parental cells); M, 100 bp DNA ladder; M1, 1 kb DNA ladder; E2, E3, G1, G9, H2, J4, B16, A20, genomic DNA from puromycin resistant and GFP-positive hESC clones obtained through co-transfection of pattP4X-PGKssPuro-UTF1-eGFP and pEF1α-ss-Int-C3CNLS . ( C ) Southern blot analysis. Genomic DNA from three hESC clones and parental hESC cells were digested with NdeI or XbaI. Digoxigenin-labeled probe to EGFP was employed. Lanes: M1, 1 kb DNA ladder; 10 8 , 10 9 , copies of linearized targeting vector as positive control; ES, parental DNA; A3, E3 and K3, genomic DNA from targeted hESC clones. White arrow heads indicate fragments of the expected size and black arrow heads indicate extra or unexpected fragments in the targeted clones. ( D ) Functional test for UTF1 promoter-driven EGFP expression in targeted hESC clones. Fluorescence microscopic analysis of undifferentiated and RA-induced, differentiated parental hES-047 cells and clones A3, E3 and K3. EGFP expression was detected with the undifferentiated hESC clones A3, E3 and K3 (column 2, panels 2, 3 and 4) but disappeared in differentiated progenies (column 4, panels 2, 3 and 4) respectively. Panels in columns 1 and 3 are phase-contrast light micrographs of undifferentiated and differentiated cells, respectively. Magnification 5×; Scale bars 100 μm. ( E ) FACS analysis for undifferentiated and differentiated hESCs clones Dot plots representing GFP + cells (upper right quadrant) and GFP − cells (lower right quadrant) for the untargeted hESCs, undifferentiated targeted hESC clones (A3, E3, K3) after 3 weeks (early) and 8 weeks (late) of culturing the cells (left and middle panel) and their differentiated progenies (right panel).
    Figure Legend Snippet: Targeting att H4X in hESCs with pattP4X-PGKssPuro-UTF1-eGFP . ( A ) Schematic diagram of pattP4X-PGKssPuro-UTF1-eGFP targeting vector after integration. Locations of primers (Puro rev24, PGK rev, pr111 and pr22) and the Southern probe are indicated. ( B ) Screening for att H4X x att P4X recombination events in selected hESC clones. Semi-nested PCR with primers cs_ att H4X_F1 and PGK rev (for the left junction) using templates obtained with primary PCR (primers cs_ att H4X_F1 and Puro rev24). PCR products of the expected size (∼900 bp) were detected in hESC clone E3 (left panel). Confirmatory PCR with genomic locus specific primers were performed for clone E3. PCR products of expected size (∼1100 bp) were obtained in a semi-nested PCR with primer PGK rev and genomic locus-specific forward primer E3-GF2 using templates from a primary PCR (primers Puro rev24 and primer E3-GF2; middle panel). PCR products of expected size (∼1000 bp) were obtained in a semi-nested PCR with primer pr111 and genomic locus specific reverse primer E3-GR2 (for the right junction) using templates from a primary PCR (primers pr22 and E3-GR2; right panel). W, no DNA template control; ES, negative control (genomic DNA from parental cells); M, 100 bp DNA ladder; M1, 1 kb DNA ladder; E2, E3, G1, G9, H2, J4, B16, A20, genomic DNA from puromycin resistant and GFP-positive hESC clones obtained through co-transfection of pattP4X-PGKssPuro-UTF1-eGFP and pEF1α-ss-Int-C3CNLS . ( C ) Southern blot analysis. Genomic DNA from three hESC clones and parental hESC cells were digested with NdeI or XbaI. Digoxigenin-labeled probe to EGFP was employed. Lanes: M1, 1 kb DNA ladder; 10 8 , 10 9 , copies of linearized targeting vector as positive control; ES, parental DNA; A3, E3 and K3, genomic DNA from targeted hESC clones. White arrow heads indicate fragments of the expected size and black arrow heads indicate extra or unexpected fragments in the targeted clones. ( D ) Functional test for UTF1 promoter-driven EGFP expression in targeted hESC clones. Fluorescence microscopic analysis of undifferentiated and RA-induced, differentiated parental hES-047 cells and clones A3, E3 and K3. EGFP expression was detected with the undifferentiated hESC clones A3, E3 and K3 (column 2, panels 2, 3 and 4) but disappeared in differentiated progenies (column 4, panels 2, 3 and 4) respectively. Panels in columns 1 and 3 are phase-contrast light micrographs of undifferentiated and differentiated cells, respectively. Magnification 5×; Scale bars 100 μm. ( E ) FACS analysis for undifferentiated and differentiated hESCs clones Dot plots representing GFP + cells (upper right quadrant) and GFP − cells (lower right quadrant) for the untargeted hESCs, undifferentiated targeted hESC clones (A3, E3, K3) after 3 weeks (early) and 8 weeks (late) of culturing the cells (left and middle panel) and their differentiated progenies (right panel).

    Techniques Used: Plasmid Preparation, Clone Assay, Nested PCR, Polymerase Chain Reaction, Negative Control, Cotransfection, Southern Blot, Labeling, Positive Control, Functional Assay, Expressing, Fluorescence, FACS

    att H4X targeting in human embryonic stem cell (hESCs). ( A ) Schematic diagram of pTZ-attP4X-UN-EF1α-eGFP targeting vector after integration into att H4X. Positions of relevant primers, the Southern probe targeting EGFP and HindIII and XbaI restriction sites are indicated. ( B ) Western blot showing Integrase expression in hESCs. Lysates from hESCs transfected with plasmids expressing Int-C3CNLS ( pCMVssInt-C3C ), 6xHIS-tagged Int-C3CNLS ( pCMVssInt-C3C-H, pEF-Int-C3C-H, pEFssInt-C3C-H ) and untransfected control cells were analyzed by western blotting with an anti-HIS tag antibody (top panel). Purified HIS-tagged Integrase C3 was employed as positive control. β-actin was used as loading control (bottom panel). ( C ) Example of screening for att H4X × att P4X recombination events in hESCs. PCR was performed with genomic DNA (extracted from neomycin-resistant, EGFP-positive hESC recombinants) and primers cs_ att H4X_F2 and att P rev (for the left junction; top left panel) and cs_ att H4X_R2 and pr21 (for the right junction; bottom left panel). PCR amplified products of the expected sizes (278 and 439 bp) were detected in clone #24. The right panel shows a PCR analysis to confirm site-specific recombination in clone #24 using different genomic locus-specific primers. PCR-amplified products of the expected sizes (∼1.25 kb with primers att P rev and 24G-F2, and ∼750 bp with primers pr21 and 24G-R1) were obtained and confirmed by sequencing. W, no DNA template control; ES, negative control (genomic DNA from parental hESCs); +, positive control (genomic DNA from HT1080 clone #19); M, 100 bp DNA ladder; M1, 1 kb DNA ladder; 16 to 27, genomic DNA from neomycin resistant hESC clones obtained through co-transfection of pTZ-attP4X-UN-EF1α-eGFP and pEF1α-ssInt-C3CNLS . ( D ) Southern blot analysis. Genomic DNA purified from three targeted hESC clones and parental hESC cell lines were digested with HindIII or XbaI. A probe complementary to EGFP was employed. Lanes: M1, 1 kb DNA ladder; m, DNA ladder (TeloTAGGG Telomere Length Assay kit, Roche); ES, parental DNA; 3, 24, 59, genomic DNA from targeted hESC clones; pUN4X (10 7 , 10 8 ), copies of linearized targeting vector pTZ-attP4X-UN-EF1α-eGFP . White arrow heads indicate fragments of the expected size in the targeted clones.
    Figure Legend Snippet: att H4X targeting in human embryonic stem cell (hESCs). ( A ) Schematic diagram of pTZ-attP4X-UN-EF1α-eGFP targeting vector after integration into att H4X. Positions of relevant primers, the Southern probe targeting EGFP and HindIII and XbaI restriction sites are indicated. ( B ) Western blot showing Integrase expression in hESCs. Lysates from hESCs transfected with plasmids expressing Int-C3CNLS ( pCMVssInt-C3C ), 6xHIS-tagged Int-C3CNLS ( pCMVssInt-C3C-H, pEF-Int-C3C-H, pEFssInt-C3C-H ) and untransfected control cells were analyzed by western blotting with an anti-HIS tag antibody (top panel). Purified HIS-tagged Integrase C3 was employed as positive control. β-actin was used as loading control (bottom panel). ( C ) Example of screening for att H4X × att P4X recombination events in hESCs. PCR was performed with genomic DNA (extracted from neomycin-resistant, EGFP-positive hESC recombinants) and primers cs_ att H4X_F2 and att P rev (for the left junction; top left panel) and cs_ att H4X_R2 and pr21 (for the right junction; bottom left panel). PCR amplified products of the expected sizes (278 and 439 bp) were detected in clone #24. The right panel shows a PCR analysis to confirm site-specific recombination in clone #24 using different genomic locus-specific primers. PCR-amplified products of the expected sizes (∼1.25 kb with primers att P rev and 24G-F2, and ∼750 bp with primers pr21 and 24G-R1) were obtained and confirmed by sequencing. W, no DNA template control; ES, negative control (genomic DNA from parental hESCs); +, positive control (genomic DNA from HT1080 clone #19); M, 100 bp DNA ladder; M1, 1 kb DNA ladder; 16 to 27, genomic DNA from neomycin resistant hESC clones obtained through co-transfection of pTZ-attP4X-UN-EF1α-eGFP and pEF1α-ssInt-C3CNLS . ( D ) Southern blot analysis. Genomic DNA purified from three targeted hESC clones and parental hESC cell lines were digested with HindIII or XbaI. A probe complementary to EGFP was employed. Lanes: M1, 1 kb DNA ladder; m, DNA ladder (TeloTAGGG Telomere Length Assay kit, Roche); ES, parental DNA; 3, 24, 59, genomic DNA from targeted hESC clones; pUN4X (10 7 , 10 8 ), copies of linearized targeting vector pTZ-attP4X-UN-EF1α-eGFP . White arrow heads indicate fragments of the expected size in the targeted clones.

    Techniques Used: Plasmid Preparation, Western Blot, Expressing, Transfection, Purification, Positive Control, Polymerase Chain Reaction, Amplification, Sequencing, Negative Control, Clone Assay, Cotransfection, Southern Blot

    22) Product Images from "A flow extension tethered particle motion assay for single-molecule proteolysis"

    Article Title: A flow extension tethered particle motion assay for single-molecule proteolysis

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.9b00106

    Scheme showing the route used to prepare DNA-conjugated peptide substrates for single-molecule proteolysis. A. Synthetic route to the double oligonucleotide-coupled peptide conjugate. Product 1 is a polyglycine conjugate made with a fluoresceinated lysine residue and a C-terminal thiol (green), coupled to a 5’-maleimide-oligo (magenta). Product 2 is the Sortase A-catalyzed conjugation of the substrate peptide containing a C-terminal LPXTG sortase acceptor motif (red) to the polyglycine-oligo (green-magenta). Product 3 (boxed) is the double oligonucleotide peptide conjugate made from linking the N-terminal cysteine of 2 to the 5’-maleimide-oligo (cyan). B. Synthesis of the digoxigenin-peptide-biotin substrate. Product 4 (boxed) is produced by an annealing and ligation reaction of a 5’ digoxigenin-containing oligonucleotide, a short (100 bp) DNA duplex and the N-terminal oligonucleotide end of the double oligo-peptide conjugate, along with simultaneous ligation of the C-terminal-end oligonucleotide to the XbaI-cleaved 24.0 kbp fragment of phage λ, and a terminal biotinylated oligonucleotide.
    Figure Legend Snippet: Scheme showing the route used to prepare DNA-conjugated peptide substrates for single-molecule proteolysis. A. Synthetic route to the double oligonucleotide-coupled peptide conjugate. Product 1 is a polyglycine conjugate made with a fluoresceinated lysine residue and a C-terminal thiol (green), coupled to a 5’-maleimide-oligo (magenta). Product 2 is the Sortase A-catalyzed conjugation of the substrate peptide containing a C-terminal LPXTG sortase acceptor motif (red) to the polyglycine-oligo (green-magenta). Product 3 (boxed) is the double oligonucleotide peptide conjugate made from linking the N-terminal cysteine of 2 to the 5’-maleimide-oligo (cyan). B. Synthesis of the digoxigenin-peptide-biotin substrate. Product 4 (boxed) is produced by an annealing and ligation reaction of a 5’ digoxigenin-containing oligonucleotide, a short (100 bp) DNA duplex and the N-terminal oligonucleotide end of the double oligo-peptide conjugate, along with simultaneous ligation of the C-terminal-end oligonucleotide to the XbaI-cleaved 24.0 kbp fragment of phage λ, and a terminal biotinylated oligonucleotide.

    Techniques Used: Conjugation Assay, Produced, Ligation

    23) Product Images from "β-nicotinamide mononucleotide (NMN) production in Escherichia coli"

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-30792-0

    pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in pUC57-kan vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with NcoI and XbaI restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.
    Figure Legend Snippet: pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in pUC57-kan vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with NcoI and XbaI restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.

    Techniques Used: Plasmid Preparation, Mutagenesis, Clone Assay, Polymerase Chain Reaction, Amplification, Purification, Construct, Transformation Assay, Agarose Gel Electrophoresis, Fluorimetry Assay

    24) Product Images from "Rapid Microarray-Based Genotyping of Enterohemorrhagic Escherichia coli Serotype O156:H25/H-/Hnt Isolates from Cattle and Clonal Relationship Analysis ▿ Serotype O156:H25/H-/Hnt Isolates from Cattle and Clonal Relationship Analysis ▿ †"

    Article Title: Rapid Microarray-Based Genotyping of Enterohemorrhagic Escherichia coli Serotype O156:H25/H-/Hnt Isolates from Cattle and Clonal Relationship Analysis ▿ Serotype O156:H25/H-/Hnt Isolates from Cattle and Clonal Relationship Analysis ▿ †

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00743-10

    Neighbor-joining tree of bovine E. coli O156 isolates based on the restriction pattern obtained after digestion with XbaI, NotI, BlnI, and SpeI (see Fig. S1 in the supplemental material).
    Figure Legend Snippet: Neighbor-joining tree of bovine E. coli O156 isolates based on the restriction pattern obtained after digestion with XbaI, NotI, BlnI, and SpeI (see Fig. S1 in the supplemental material).

    Techniques Used:

    25) Product Images from "The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining ▿The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining ▿ †"

    Article Title: The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining ▿The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.05849-11

    In vitro NHEJ assay using DNA substrate with noncohesive ends. (A) Schematic showing the derivation of the 5.7-kb fragment with noncohesive BstXI termini, A and B. Corresponding PCR primers a and b contain HindIII and XbaI sites, respectively. Possible
    Figure Legend Snippet: In vitro NHEJ assay using DNA substrate with noncohesive ends. (A) Schematic showing the derivation of the 5.7-kb fragment with noncohesive BstXI termini, A and B. Corresponding PCR primers a and b contain HindIII and XbaI sites, respectively. Possible

    Techniques Used: In Vitro, Non-Homologous End Joining, Polymerase Chain Reaction

    26) Product Images from "RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases"

    Article Title: RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases

    Journal: Genome Research

    doi: 10.1101/gr.219394.116

    Analysis of restriction sites over genic and intergenic regions. ( A ) Restriction fragment lengths over genic regions (gene bodies, exons, first exons) are significantly larger compared to intergenic regions. The plot shows the difference of genic (observed) and intergenic (expected) fragment sizes in base pairs. The following enzymes were applied in combination: HindIII, EcoRI, BsrGI, XbaI, and SspI. ( B – D ) The number of restriction sites over genic regions is significantly lower compared to intergenic regions. Colors indicate the proportion of cutting sites in each category. Red and blue slices, marking the rarest restriction site frequencies, are prevalent over genic elements in each pie chart. ( E ) Cutting efficiency of restriction enzymes applied in the indicated DRIP-seq experiments. Zero read: the restriction site was cut. Greater equal than one read: the restriction site was uncut in a fraction of cells. There were uncut reads (sites) over half of the theoretical restriction sites. The proportion of uncut reads was even higher within gene coding regions compared to intergenic regions. See the model of cutting efficiency in panel F .
    Figure Legend Snippet: Analysis of restriction sites over genic and intergenic regions. ( A ) Restriction fragment lengths over genic regions (gene bodies, exons, first exons) are significantly larger compared to intergenic regions. The plot shows the difference of genic (observed) and intergenic (expected) fragment sizes in base pairs. The following enzymes were applied in combination: HindIII, EcoRI, BsrGI, XbaI, and SspI. ( B – D ) The number of restriction sites over genic regions is significantly lower compared to intergenic regions. Colors indicate the proportion of cutting sites in each category. Red and blue slices, marking the rarest restriction site frequencies, are prevalent over genic elements in each pie chart. ( E ) Cutting efficiency of restriction enzymes applied in the indicated DRIP-seq experiments. Zero read: the restriction site was cut. Greater equal than one read: the restriction site was uncut in a fraction of cells. There were uncut reads (sites) over half of the theoretical restriction sites. The proportion of uncut reads was even higher within gene coding regions compared to intergenic regions. See the model of cutting efficiency in panel F .

    Techniques Used:

    Experimental design: constructing DRIP schemes. ( A ) Experiments 1–16 explore the effect of formaldehyde-fixation (Step 1), nucleic acid isolation (Step 2), removal of free RNA (Step 3), and nucleic acid fragmentation (Step 4) on the outcome of RNA-DNA hybrid detection. Each experiment was performed at two parallel cell lysis temperatures (65°C and 37°C), respectively. The temperature variable is not depicted in the cartoon, but it is referred in the main text. ( B ) Experiments 17–24 test the impact of acoustic sharing performed on a chromatin prep rather than on naked nucleic acid, similarly to the ChIP protocol. Each experiment was performed at 65°C cell lysis temperature. ( C ) Workflow of a ChIP experiment (shown only for comparison with the DRIP pipeline). (HCHO) Formaldehyde fixation, (Phe/Chl) phenol-chloroform extraction, (Kit) silica membrane-based nucleic acid purification, (RNase A) Ribonuclease A digestion performed at high (300 mM) NaCl concentration, (Son) sonication, (RE) restriction enzyme cocktail digestion (HindIII, EcoRI, BsrGI, XbaI, and SspI). As a negative control, RNase H digestion was applied in all DRIP experiments (not indicated in the cartoon).
    Figure Legend Snippet: Experimental design: constructing DRIP schemes. ( A ) Experiments 1–16 explore the effect of formaldehyde-fixation (Step 1), nucleic acid isolation (Step 2), removal of free RNA (Step 3), and nucleic acid fragmentation (Step 4) on the outcome of RNA-DNA hybrid detection. Each experiment was performed at two parallel cell lysis temperatures (65°C and 37°C), respectively. The temperature variable is not depicted in the cartoon, but it is referred in the main text. ( B ) Experiments 17–24 test the impact of acoustic sharing performed on a chromatin prep rather than on naked nucleic acid, similarly to the ChIP protocol. Each experiment was performed at 65°C cell lysis temperature. ( C ) Workflow of a ChIP experiment (shown only for comparison with the DRIP pipeline). (HCHO) Formaldehyde fixation, (Phe/Chl) phenol-chloroform extraction, (Kit) silica membrane-based nucleic acid purification, (RNase A) Ribonuclease A digestion performed at high (300 mM) NaCl concentration, (Son) sonication, (RE) restriction enzyme cocktail digestion (HindIII, EcoRI, BsrGI, XbaI, and SspI). As a negative control, RNase H digestion was applied in all DRIP experiments (not indicated in the cartoon).

    Techniques Used: Isolation, Lysis, Chromatin Immunoprecipitation, Nucleic Acid Purification, Concentration Assay, Sonication, Negative Control

    Large restriction fragments over gene bodies cause uncertainty in the precise localization of R-loops, potentially impeding their functional annotation. ( A – C ) Genome browser tracks showing three representative examples ( MYC , BCL6 , and VIM ). Upper two tracks: restriction fragment-sized R-loops are prevalent over the 5′ end of genes, vastly exceeding the gene borders in the case of MYC . Lower two tracks: the precise genomic position of R-loops was resolved in the sonicated group of samples. Green boxes represent R-loop enriched regions predicted by the peak callers. Blue dashed lines represent cutting sites for restriction enzymes (HindIII, EcoRI, BsrGI, XbaI, and SspI).
    Figure Legend Snippet: Large restriction fragments over gene bodies cause uncertainty in the precise localization of R-loops, potentially impeding their functional annotation. ( A – C ) Genome browser tracks showing three representative examples ( MYC , BCL6 , and VIM ). Upper two tracks: restriction fragment-sized R-loops are prevalent over the 5′ end of genes, vastly exceeding the gene borders in the case of MYC . Lower two tracks: the precise genomic position of R-loops was resolved in the sonicated group of samples. Green boxes represent R-loop enriched regions predicted by the peak callers. Blue dashed lines represent cutting sites for restriction enzymes (HindIII, EcoRI, BsrGI, XbaI, and SspI).

    Techniques Used: Functional Assay, Sonication

    27) Product Images from "Generation of an Oncolytic Herpes Simplex Virus 1 Expressing Human MelanA"

    Article Title: Generation of an Oncolytic Herpes Simplex Virus 1 Expressing Human MelanA

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00002

    Generation of HSV-1 d 106S-MelanA. (A) Agarose gel image of full-length MelanA amplified from expression plasmid pcDNA3(+) MART-1 ( 29 ) using XbaI/NheI-containing primers. (B) Light microscopic images of uninfected E11 cells (left) and cytopathic effects induced in these cells 56 h post HSV-1 d 106S infection (right). (C) EcoRI digestion of HSV-1 d 106S DNA obtained from viral nucleocapsids showing distinct bands as evidence of DNA integrity. (D) Overlay of phase contrast and immunofluorescence microscopy of E11 cells harboring fluorescing (upper part) and non-fluorescing (lower part) viral plaques, representing HSV-1 d 106S and HSV-1 d 106S-MelanA, respectively, after cotransfection of the linearized transfer plasmid pd27B-MelanA and HSV-1 d 106S DNA. Light and immunofluorescence microscopy were taken using the DMI 6000B inverted microscope (20 × magnification).
    Figure Legend Snippet: Generation of HSV-1 d 106S-MelanA. (A) Agarose gel image of full-length MelanA amplified from expression plasmid pcDNA3(+) MART-1 ( 29 ) using XbaI/NheI-containing primers. (B) Light microscopic images of uninfected E11 cells (left) and cytopathic effects induced in these cells 56 h post HSV-1 d 106S infection (right). (C) EcoRI digestion of HSV-1 d 106S DNA obtained from viral nucleocapsids showing distinct bands as evidence of DNA integrity. (D) Overlay of phase contrast and immunofluorescence microscopy of E11 cells harboring fluorescing (upper part) and non-fluorescing (lower part) viral plaques, representing HSV-1 d 106S and HSV-1 d 106S-MelanA, respectively, after cotransfection of the linearized transfer plasmid pd27B-MelanA and HSV-1 d 106S DNA. Light and immunofluorescence microscopy were taken using the DMI 6000B inverted microscope (20 × magnification).

    Techniques Used: Agarose Gel Electrophoresis, Amplification, Expressing, Plasmid Preparation, Infection, Immunofluorescence, Microscopy, Cotransfection, Inverted Microscopy

    28) Product Images from "β-nicotinamide mononucleotide (NMN) production in Escherichia coli"

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-30792-0

    pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in pUC57-kan vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with NcoI and XbaI restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.
    Figure Legend Snippet: pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in pUC57-kan vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with NcoI and XbaI restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.

    Techniques Used: Plasmid Preparation, Mutagenesis, Clone Assay, Polymerase Chain Reaction, Amplification, Purification, Construct, Transformation Assay, Agarose Gel Electrophoresis, Fluorimetry Assay

    29) Product Images from "CTAG-Containing Cleavage Site Profiling to Delineate Salmonella into Natural Clusters"

    Article Title: CTAG-Containing Cleavage Site Profiling to Delineate Salmonella into Natural Clusters

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0103388

    Physical map comparison between S. gallinarum strains 287/91 and SARB21. The map of SARB21 was reported previously [21] ; here letter designations for the cleavage fragments of SARB21 have been changed according to the homologues in strain 287/91 for the convenience of comparison. Note that all XbaI, I-CeuI and AvrII (maps from top to bottom) cleavage sites are conserved in the two strains except the AvrII site between fragments F and J in 287/91 (open arrow), which is missing from SARB21. Lines with solid arrowheads at both ends indicate the ranges of genomic inversions via rrn -mediated recombination between the two strains and filled arrows indicate recombination sites that have resulted in the translocation of I-CeuI fragment D.
    Figure Legend Snippet: Physical map comparison between S. gallinarum strains 287/91 and SARB21. The map of SARB21 was reported previously [21] ; here letter designations for the cleavage fragments of SARB21 have been changed according to the homologues in strain 287/91 for the convenience of comparison. Note that all XbaI, I-CeuI and AvrII (maps from top to bottom) cleavage sites are conserved in the two strains except the AvrII site between fragments F and J in 287/91 (open arrow), which is missing from SARB21. Lines with solid arrowheads at both ends indicate the ranges of genomic inversions via rrn -mediated recombination between the two strains and filled arrows indicate recombination sites that have resulted in the translocation of I-CeuI fragment D.

    Techniques Used: Translocation Assay

    XbaI and AvrII cleavage patterns of S. gallinarum strains 287/91 and SARB21 after PFGE separation. (A) XbaI cleavage. Lanes: 1, SARB21; 2, 287/91; 3, λDNA as molecular size marker. (B) AvrII cleavage. Lanes: 1, λDNA as molecular size marker; 2, SARB21; 3, 287/91. Letter designations are for strain 287/91; the same letters are used for homologous fragments in strain SARB21. In the designation of fragments in SARB21, C′ means a fragment homologous to C in 287/91 but truncated on the right-hand part by genomic rearrangement, and ‘C means truncation on the left-hand part of the fragment.
    Figure Legend Snippet: XbaI and AvrII cleavage patterns of S. gallinarum strains 287/91 and SARB21 after PFGE separation. (A) XbaI cleavage. Lanes: 1, SARB21; 2, 287/91; 3, λDNA as molecular size marker. (B) AvrII cleavage. Lanes: 1, λDNA as molecular size marker; 2, SARB21; 3, 287/91. Letter designations are for strain 287/91; the same letters are used for homologous fragments in strain SARB21. In the designation of fragments in SARB21, C′ means a fragment homologous to C in 287/91 but truncated on the right-hand part by genomic rearrangement, and ‘C means truncation on the left-hand part of the fragment.

    Techniques Used: Marker

    30) Product Images from "A flow extension tethered particle motion assay for single-molecule proteolysis"

    Article Title: A flow extension tethered particle motion assay for single-molecule proteolysis

    Journal: bioRxiv

    doi: 10.1101/528919

    Scheme showing the route used to prepare DNA-conjugated peptide substrates for single-molecule proteolysis. A. Synthetic route to the double oligonucleotide-coupled peptide conjugate. Product 1 is a polyglycine conjugate made with a fluoresceinated lysine residue and a C-terminal thiol (green), coupled to a 5’-maleimide-oligo (magenta). Product 2 is the Sortase A-catalyzed conjugation of the substrate peptide containing a C-terminal LPXTG sortase acceptor motif (red) to the polyglycine-oligo (green-magenta). Product 3 (boxed) is the double oligonucleotide peptide conjugate made from linking the N-terminal cysteine of 2 to the 5’-maleimide-oligo (cyan). B. Synthesis of the digoxigenin-peptide-biotin substrate. Product 4 (boxed) is produced by an annealing and ligation reaction of a 5’ digoxigenin-containing oligonucleotide, a short (100 bp) DNA duplex and the N-terminal oligonucleotide end of the double oligo-peptide conjugate, along with simultaneous ligation of the C-terminal-end oligonucleotide to the XbaI-cleaved 24.0 kbp fragment of phage λ, and a terminal biotinylated oligonucleotide.
    Figure Legend Snippet: Scheme showing the route used to prepare DNA-conjugated peptide substrates for single-molecule proteolysis. A. Synthetic route to the double oligonucleotide-coupled peptide conjugate. Product 1 is a polyglycine conjugate made with a fluoresceinated lysine residue and a C-terminal thiol (green), coupled to a 5’-maleimide-oligo (magenta). Product 2 is the Sortase A-catalyzed conjugation of the substrate peptide containing a C-terminal LPXTG sortase acceptor motif (red) to the polyglycine-oligo (green-magenta). Product 3 (boxed) is the double oligonucleotide peptide conjugate made from linking the N-terminal cysteine of 2 to the 5’-maleimide-oligo (cyan). B. Synthesis of the digoxigenin-peptide-biotin substrate. Product 4 (boxed) is produced by an annealing and ligation reaction of a 5’ digoxigenin-containing oligonucleotide, a short (100 bp) DNA duplex and the N-terminal oligonucleotide end of the double oligo-peptide conjugate, along with simultaneous ligation of the C-terminal-end oligonucleotide to the XbaI-cleaved 24.0 kbp fragment of phage λ, and a terminal biotinylated oligonucleotide.

    Techniques Used: Conjugation Assay, Produced, Ligation

    Scheme for assembly of DNA-only control substrate. Product (5) (boxed) is produced by annealing and ligation of a 5’ digoxigenin-containing oligonucleotide, a short (100 bp) DNA duplex, a bridging oligonucleotide (oligonucleotide 8, cyan and black), the XbaI-cleaved 24.5 kbp fragment of phage λ, and a terminal biotinylated oligonucleotide.
    Figure Legend Snippet: Scheme for assembly of DNA-only control substrate. Product (5) (boxed) is produced by annealing and ligation of a 5’ digoxigenin-containing oligonucleotide, a short (100 bp) DNA duplex, a bridging oligonucleotide (oligonucleotide 8, cyan and black), the XbaI-cleaved 24.5 kbp fragment of phage λ, and a terminal biotinylated oligonucleotide.

    Techniques Used: Produced, Ligation

    31) Product Images from "The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining ▿The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining ▿ †"

    Article Title: The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining ▿The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.05849-11

    In vitro NHEJ assay using DNA substrate with noncohesive ends. (A) Schematic showing the derivation of the 5.7-kb fragment with noncohesive BstXI termini, A and B. Corresponding PCR primers a and b contain HindIII and XbaI sites, respectively. Possible
    Figure Legend Snippet: In vitro NHEJ assay using DNA substrate with noncohesive ends. (A) Schematic showing the derivation of the 5.7-kb fragment with noncohesive BstXI termini, A and B. Corresponding PCR primers a and b contain HindIII and XbaI sites, respectively. Possible

    Techniques Used: In Vitro, Non-Homologous End Joining, Polymerase Chain Reaction

    32) Product Images from "Association of iss and iucA, but Not tsh, with Plasmid-Mediated Virulence of Avian Pathogenic Escherichia coli"

    Article Title: Association of iss and iucA, but Not tsh, with Plasmid-Mediated Virulence of Avian Pathogenic Escherichia coli

    Journal:

    doi: 10.1128/IAI.72.11.6554-6560.2004

    Physical map of plasmid pVM01::Tn phoA illustrating the NotI, SpeI, and XbaI restriction endonuclease cleavage sites and the locations of the Tn phoA insert and the putative virulence genes, iucA , iss , and tsh. Designation of fragments (A,B,C, or D) corresponds
    Figure Legend Snippet: Physical map of plasmid pVM01::Tn phoA illustrating the NotI, SpeI, and XbaI restriction endonuclease cleavage sites and the locations of the Tn phoA insert and the putative virulence genes, iucA , iss , and tsh. Designation of fragments (A,B,C, or D) corresponds

    Techniques Used: Plasmid Preparation

    33) Product Images from "The SMC5/6 Complex Interacts with the Papillomavirus E2 Protein and Influences Maintenance of Viral Episomal DNA"

    Article Title: The SMC5/6 Complex Interacts with the Papillomavirus E2 Protein and Influences Maintenance of Viral Episomal DNA

    Journal: Journal of Virology

    doi: 10.1128/JVI.00356-18

    SMC6 is not required for differentiation-dependent amplification of viral DNA. CIN612-9E cells were lentivirally transduced with control or SMC6 shRNA on day 0. Uninfected cells were collected at day 0. At 3 days postinfection, undifferentiated cells were harvested for analysis by qRT-PCR and Southern blotting or underwent 96 h of differentiation in high-calcium medium. (A) qRT-PCR measured involucrin levels as a marker of keratinocyte differentiation. (B) SMC6 mRNA levels were measured by qRT-PCR in CIN612-9E cells lentivirally transduced with SMC6 shRNA or GFP shRNA. All qRT-PCR data are normalized to those for cyclophilin A ( n = 4). (C) (Left) Southern blotting detected HPV31 DNA from CIN612-9E cells. (Top) Total DNA was digested with BamHI targeting only mammalian DNA for visualization of the various forms of viral DNA. (Middle) Alternatively, DNA was digested with XbaI to linearize viral DNA (8 kbp). (Bottom) The Southern blot containing XbaI-digested DNA was probed for the human tPa gene as a loading control (1 to 2 kbp). (Right) Densitometric analysis of the episomal form of DNA as visualized by Southern blotting. P values were calculated using Student's t test. ***, P
    Figure Legend Snippet: SMC6 is not required for differentiation-dependent amplification of viral DNA. CIN612-9E cells were lentivirally transduced with control or SMC6 shRNA on day 0. Uninfected cells were collected at day 0. At 3 days postinfection, undifferentiated cells were harvested for analysis by qRT-PCR and Southern blotting or underwent 96 h of differentiation in high-calcium medium. (A) qRT-PCR measured involucrin levels as a marker of keratinocyte differentiation. (B) SMC6 mRNA levels were measured by qRT-PCR in CIN612-9E cells lentivirally transduced with SMC6 shRNA or GFP shRNA. All qRT-PCR data are normalized to those for cyclophilin A ( n = 4). (C) (Left) Southern blotting detected HPV31 DNA from CIN612-9E cells. (Top) Total DNA was digested with BamHI targeting only mammalian DNA for visualization of the various forms of viral DNA. (Middle) Alternatively, DNA was digested with XbaI to linearize viral DNA (8 kbp). (Bottom) The Southern blot containing XbaI-digested DNA was probed for the human tPa gene as a loading control (1 to 2 kbp). (Right) Densitometric analysis of the episomal form of DNA as visualized by Southern blotting. P values were calculated using Student's t test. ***, P

    Techniques Used: Amplification, Transduction, shRNA, Quantitative RT-PCR, Southern Blot, Marker

    34) Product Images from "Acinetobacter baumannii Gastrointestinal Colonization Is Facilitated by Secretory IgA Which Is Reductively Dissociated by Bacterial Thioredoxin A"

    Article Title: Acinetobacter baumannii Gastrointestinal Colonization Is Facilitated by Secretory IgA Which Is Reductively Dissociated by Bacterial Thioredoxin A

    Journal: mBio

    doi: 10.1128/mBio.01298-18

    Generation of a trxA deletion mutant. A schematic representation of the WT A. baumannii Ci79 and Δ trxA mutant genome surrounding the trxA locus with predicted fragment sizes detected by probe following either HindIII (white arrowhead) or XbaI (black arrowhead) digestion as well as probe target region (dotted box) (A). Southern blot (B) and Western blot (C) analyses of genomic and protein extracts, respectively, from WT Ci79, ΔtrxA , and ΔtrxA c A. baumannii were subsequently performed. PCR amplification of the trxA gene locus was performed using DNA from WT Ci79, ΔtrxA , and ΔtrxA c A. baumannii isolates. The PCR amplicons were subsequently digested with SalI and subjected to agarose gel electrophoresis (C). Total bacterial proteins were separated by electrophoresis and visualized in a Coomassie blue-stained polyacrylamide gel (D, left panel) or probed with anti-TrxA antibody to detect TrxA expression (D, right panel). Molecular marker sizes for DNA (bp; B and C) and protein (kDa; D) are provided.
    Figure Legend Snippet: Generation of a trxA deletion mutant. A schematic representation of the WT A. baumannii Ci79 and Δ trxA mutant genome surrounding the trxA locus with predicted fragment sizes detected by probe following either HindIII (white arrowhead) or XbaI (black arrowhead) digestion as well as probe target region (dotted box) (A). Southern blot (B) and Western blot (C) analyses of genomic and protein extracts, respectively, from WT Ci79, ΔtrxA , and ΔtrxA c A. baumannii were subsequently performed. PCR amplification of the trxA gene locus was performed using DNA from WT Ci79, ΔtrxA , and ΔtrxA c A. baumannii isolates. The PCR amplicons were subsequently digested with SalI and subjected to agarose gel electrophoresis (C). Total bacterial proteins were separated by electrophoresis and visualized in a Coomassie blue-stained polyacrylamide gel (D, left panel) or probed with anti-TrxA antibody to detect TrxA expression (D, right panel). Molecular marker sizes for DNA (bp; B and C) and protein (kDa; D) are provided.

    Techniques Used: Mutagenesis, Southern Blot, Western Blot, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Electrophoresis, Staining, Expressing, Marker

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    Clone Assay:

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    Article Snippet: .. The PCR product was purified using QIAquick Gel Extraction Kit (Qiagen), digested with Hind III (NEB) and Xba I (NEB), and cloned into the pUC119 plasmid vector. .. Approximately 30 plasmid clones for each sorted sIgM (−) population were sequenced using universal forward, reverse, and Ig heavy chain primers 3 and 4.

    Luciferase:

    Article Title: Construction of a Bioluminescent Labelling Plasmid Vector for Bifidobacteria
    Article Snippet: .. A luciferase gene (luc+ ) was liberated with XbaI and EcoRI restriction endonucleases (New England Biolabs) from pLuc2 recombinant plasmid and inserted into pTG262 vector. .. The cloned pFI2576 rep was digested with SalI enzyme and subcloned into recombinant pTG262 (luc+ ) digested with the same enzyme.

    Agarose Gel Electrophoresis:

    Article Title: Antimicrobial Resistance, Virulence Profiles and Molecular Subtypes of Salmonella enterica Serovars Typhi and Paratyphi A Blood Isolates from Kolkata, India during 2009-2013
    Article Snippet: .. The DNA plugs were digested with 40 U of Xba I (New England Biolabs, MA) at 37°C for 18 h. The digested DNA was run on 1% pulsed field certified agarose gel (Bio-Rad, Hercules, Calif.) prepared in 0.5x TBE buffer (Sigma) using CHEF DRIII (Bio-Rad) apparatus with an initial switch time of 2.2 sec and a final switch time of 63.8 sec at 6 V/cm for 24 h. The gel was stained with ethidium bromide (1 µg/ml, Sigma), destained with deionized water and PFGE profiles were observed with the UV trans-illuminator using GelDoc (Bio-Rad). ..

    Article Title: Generation and Characterization of a Transgenic Pig Carrying a DsRed-Monomer Reporter Gene
    Article Snippet: .. The DNA were digested using XbaI and DraI (New England Biolabs, Beverly, MA, USA), separated in 1% agarose gel, and then transferred to a Hybond-N+ membrane (Amersham Biosciences) by using capillary diffusion. .. The blots were probed with the 232-bp PCR fragment of DsRed (forward, 5′- CGACATCCCCGACTACATGA-3′ , and reverse, 5′- TCCTGGGGGTACAGCTTCTC-3′ ), which was [p32 ]dCTP-labeled by using the random primed labeling method.

    Article Title: Acinetobacter baumannii Gastrointestinal Colonization Is Facilitated by Secretory IgA Which Is Reductively Dissociated by Bacterial Thioredoxin A
    Article Snippet: .. Genomic DNAs from Ci79, Δ trxA , and Δ trxA c strains were isolated using the GeneJET genomic DNA purification kit (Thermo Scientific, Rockford, IL), digested with XbaI or HindIII (New England BioLabs), and run on an 0.8% agarose gel at 16 V until completion. .. DNA was transferred to a nylon Biodyne B membrane (Thermo Scientific, Rockford, IL) and UV-cross-linked for 5 min. A DNA probe amplified from WT A. baumannii Ci79 genomic DNA using the DN_Fw/DN_Rv primer set was biotin labeled using the North2South biotin random prime labeling kit (Thermo Scientific, Rockport, IL).

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    Article Snippet: .. For each plant line, 15 µg DNA was digested with 50 U Xba I (New England Biolabs, Ipswich, MA) for 16 hr at 37 ° C, then separated on a 0.8% agarose gel at 70 V for 16 hr and transferred to a GeneScreen Plus nylon membrane (PerkinElmer, Boston, MA) in 10× saline sodium citrate (SSC) buffer (1.5 M NaCl, 0.15 M sodium citrate, pH 7). .. The membrane was UV cross-linked and hybridized for 16 hr at 42 ° C with a [α-32 P] dATP labeled 16D10i-2 probe ( ) in hybridization buffer (50% deionized formamide, 0.1 mg/ml salmon sperm DNA, 1% sodium dodecyl sulfate (SDS), 1 M NaCl, 10% dextran sulfate).

    Isolation:

    Article Title: Acinetobacter baumannii Gastrointestinal Colonization Is Facilitated by Secretory IgA Which Is Reductively Dissociated by Bacterial Thioredoxin A
    Article Snippet: .. Genomic DNAs from Ci79, Δ trxA , and Δ trxA c strains were isolated using the GeneJET genomic DNA purification kit (Thermo Scientific, Rockford, IL), digested with XbaI or HindIII (New England BioLabs), and run on an 0.8% agarose gel at 16 V until completion. .. DNA was transferred to a nylon Biodyne B membrane (Thermo Scientific, Rockford, IL) and UV-cross-linked for 5 min. A DNA probe amplified from WT A. baumannii Ci79 genomic DNA using the DN_Fw/DN_Rv primer set was biotin labeled using the North2South biotin random prime labeling kit (Thermo Scientific, Rockport, IL).

    Size-exclusion Chromatography:

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    Article Snippet: .. The DNA plugs were digested with 40 U of Xba I (New England Biolabs, MA) at 37°C for 18 h. The digested DNA was run on 1% pulsed field certified agarose gel (Bio-Rad, Hercules, Calif.) prepared in 0.5x TBE buffer (Sigma) using CHEF DRIII (Bio-Rad) apparatus with an initial switch time of 2.2 sec and a final switch time of 63.8 sec at 6 V/cm for 24 h. The gel was stained with ethidium bromide (1 µg/ml, Sigma), destained with deionized water and PFGE profiles were observed with the UV trans-illuminator using GelDoc (Bio-Rad). ..

    Purification:

    Article Title: A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism
    Article Snippet: .. The PCR product was purified using QIAquick Gel Extraction Kit (Qiagen), digested with Hind III (NEB) and Xba I (NEB), and cloned into the pUC119 plasmid vector. .. Approximately 30 plasmid clones for each sorted sIgM (−) population were sequenced using universal forward, reverse, and Ig heavy chain primers 3 and 4.

    Diffusion-based Assay:

    Article Title: Generation and Characterization of a Transgenic Pig Carrying a DsRed-Monomer Reporter Gene
    Article Snippet: .. The DNA were digested using XbaI and DraI (New England Biolabs, Beverly, MA, USA), separated in 1% agarose gel, and then transferred to a Hybond-N+ membrane (Amersham Biosciences) by using capillary diffusion. .. The blots were probed with the 232-bp PCR fragment of DsRed (forward, 5′- CGACATCCCCGACTACATGA-3′ , and reverse, 5′- TCCTGGGGGTACAGCTTCTC-3′ ), which was [p32 ]dCTP-labeled by using the random primed labeling method.

    Pulsed-Field Gel:

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    DNA Purification:

    Article Title: Acinetobacter baumannii Gastrointestinal Colonization Is Facilitated by Secretory IgA Which Is Reductively Dissociated by Bacterial Thioredoxin A
    Article Snippet: .. Genomic DNAs from Ci79, Δ trxA , and Δ trxA c strains were isolated using the GeneJET genomic DNA purification kit (Thermo Scientific, Rockford, IL), digested with XbaI or HindIII (New England BioLabs), and run on an 0.8% agarose gel at 16 V until completion. .. DNA was transferred to a nylon Biodyne B membrane (Thermo Scientific, Rockford, IL) and UV-cross-linked for 5 min. A DNA probe amplified from WT A. baumannii Ci79 genomic DNA using the DN_Fw/DN_Rv primer set was biotin labeled using the North2South biotin random prime labeling kit (Thermo Scientific, Rockport, IL).

    Staining:

    Article Title: Antimicrobial Resistance, Virulence Profiles and Molecular Subtypes of Salmonella enterica Serovars Typhi and Paratyphi A Blood Isolates from Kolkata, India during 2009-2013
    Article Snippet: .. The DNA plugs were digested with 40 U of Xba I (New England Biolabs, MA) at 37°C for 18 h. The digested DNA was run on 1% pulsed field certified agarose gel (Bio-Rad, Hercules, Calif.) prepared in 0.5x TBE buffer (Sigma) using CHEF DRIII (Bio-Rad) apparatus with an initial switch time of 2.2 sec and a final switch time of 63.8 sec at 6 V/cm for 24 h. The gel was stained with ethidium bromide (1 µg/ml, Sigma), destained with deionized water and PFGE profiles were observed with the UV trans-illuminator using GelDoc (Bio-Rad). ..

    Polymerase Chain Reaction:

    Article Title: A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism
    Article Snippet: .. The PCR product was purified using QIAquick Gel Extraction Kit (Qiagen), digested with Hind III (NEB) and Xba I (NEB), and cloned into the pUC119 plasmid vector. .. Approximately 30 plasmid clones for each sorted sIgM (−) population were sequenced using universal forward, reverse, and Ig heavy chain primers 3 and 4.

    Gel Extraction:

    Article Title: A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism
    Article Snippet: .. The PCR product was purified using QIAquick Gel Extraction Kit (Qiagen), digested with Hind III (NEB) and Xba I (NEB), and cloned into the pUC119 plasmid vector. .. Approximately 30 plasmid clones for each sorted sIgM (−) population were sequenced using universal forward, reverse, and Ig heavy chain primers 3 and 4.

    Recombinant:

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    Article Snippet: .. A luciferase gene (luc+ ) was liberated with XbaI and EcoRI restriction endonucleases (New England Biolabs) from pLuc2 recombinant plasmid and inserted into pTG262 vector. .. The cloned pFI2576 rep was digested with SalI enzyme and subcloned into recombinant pTG262 (luc+ ) digested with the same enzyme.

    Plasmid Preparation:

    Article Title: A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism
    Article Snippet: .. The PCR product was purified using QIAquick Gel Extraction Kit (Qiagen), digested with Hind III (NEB) and Xba I (NEB), and cloned into the pUC119 plasmid vector. .. Approximately 30 plasmid clones for each sorted sIgM (−) population were sequenced using universal forward, reverse, and Ig heavy chain primers 3 and 4.

    Article Title: Construction of a Bioluminescent Labelling Plasmid Vector for Bifidobacteria
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    • xbai site located  (New England Biolabs)
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    New England Biolabs xbai site located
    (GAA•TTC) n constructs used to analyze effect of DSB repair on triplet-repeat instability. pUC19 based constructs are shown containing either uninterrupted (GAA•TTC) 79 or (GAA•TTC) n sequences engineered to contain a <t>XbaI</t> recognition sequence at specific locations within the repeat tract (see Materials and Methods section for details). Repeat tracts were cloned in both orientations relative to the origin of replication (arrow indicates direction of replication). Repeat-containing plasmids are depicted either in the <t>‘GAA</t> orientation’ (e.g. GAA-79) or ‘TTC’ orientation (e.g. TTC-79), based on whether (GAA) n or (TTC) n serves as the lagging strand template, respectively. Numbers within the boxes indicate the length of the uninterrupted (GAA•TTC) n sequence.
    Xbai Site Located, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 320 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    algr d54e xbai r  (New England Biolabs)
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    New England Biolabs algr d54e xbai r
    Phosphorylated <t>AlgR</t> and AlgR <t>D54E</t> bound to the fimU promoter. The effect of in vitro phosphorylation of AlgR on binding to an 82-bp fimU DNA fragment was compared with the binding of purified AlgR D54N or AlgR D54E using EMSA. (A) Diagram of the 82-bp fragment used in EMSAs and its location within the fimT-fimU intergenic region. (B) Increasing amounts of the phosphate donor, AcP, were added to the AlgR protein in order to determine the effect of phosphorylation on DNA binding. Wild-type AlgR protein was pretreated with AcP for 30 min prior to binding reactions. Lanes 1 to 8, fimU DNA alone (lane 1) or in the presence of AlgR without AcP (lane 2) or with increasing concentrations of AcP (lanes 3 to 8). Lanes 9 to 12, the AlgR-P–DNA complex was competed with the addition of nonradiolabeled fimU DNA. (C) Phosphorylated AlgR protein was compared with untreated AlgR for DNA binding. Purified AlgR protein was pretreated either with water only or with AcP (500× molar excess of protein) prior to binding reactions. Lanes 1 to 10, fimU DNA incubated with a no-protein control (lane 1), increasing concentrations of AlgR (lanes 2 to 5) or AlgR-P (lanes 6 to 9), or 5 mM AcP only (lane 10). Lanes 11 to 13, 5 nM pscEF DNA incubated with a no-protein control (lane 11), 2.5 μM AlgR (lane 12), or 2.5 μM AlgR-P (lane 13). (D) Purified AlgR D54N or AlgR D54E protein was tested for binding to fimU DNA. Lane 1, no-protein control; lanes 2 to 5, increasing concentrations of AlgR D54N; lanes 6 to 9, increasing concentrations of AlgR D54E (lanes 6 to 9). Open arrowheads, unbound DNA; filled arrowheads, AlgR-DNA complexes. All concentrations given in the figure are final binding reaction concentrations.
    Algr D54e Xbai R, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (GAA•TTC) n constructs used to analyze effect of DSB repair on triplet-repeat instability. pUC19 based constructs are shown containing either uninterrupted (GAA•TTC) 79 or (GAA•TTC) n sequences engineered to contain a XbaI recognition sequence at specific locations within the repeat tract (see Materials and Methods section for details). Repeat tracts were cloned in both orientations relative to the origin of replication (arrow indicates direction of replication). Repeat-containing plasmids are depicted either in the ‘GAA orientation’ (e.g. GAA-79) or ‘TTC’ orientation (e.g. TTC-79), based on whether (GAA) n or (TTC) n serves as the lagging strand template, respectively. Numbers within the boxes indicate the length of the uninterrupted (GAA•TTC) n sequence.

    Journal: Nucleic Acids Research

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence

    doi: 10.1093/nar/gkm1066

    Figure Lengend Snippet: (GAA•TTC) n constructs used to analyze effect of DSB repair on triplet-repeat instability. pUC19 based constructs are shown containing either uninterrupted (GAA•TTC) 79 or (GAA•TTC) n sequences engineered to contain a XbaI recognition sequence at specific locations within the repeat tract (see Materials and Methods section for details). Repeat tracts were cloned in both orientations relative to the origin of replication (arrow indicates direction of replication). Repeat-containing plasmids are depicted either in the ‘GAA orientation’ (e.g. GAA-79) or ‘TTC’ orientation (e.g. TTC-79), based on whether (GAA) n or (TTC) n serves as the lagging strand template, respectively. Numbers within the boxes indicate the length of the uninterrupted (GAA•TTC) n sequence.

    Article Snippet: The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

    Techniques: Construct, Sequencing, Clone Assay

    DSB repair mediated instability of the (GAA•TTC) n sequence is independent of RecA. ( A ) Repair of linear templates obtained by XbaI digestion of GAA-70X, TTC-70X, GAA-30X and TTC-30X resulted in similar levels of instability when transformed into E. coli MM28 [wild-type (WT)] and its isogenic recA mutant strain, M152. There was no difference in the level of repeat instability between the RecA-proficient and RecA-deficient strains, regardless of repeat length or the orientation with respect to the origin of replication. ( B ) Instability of GAA-30X constructs either uncut or following repair at the PstI, XbaI or KpnI restriction sites in isogenic E. coli strains AB1157 (RecA-proficient) and JC10287 (RecA-deficient) showing that DSB repair-mediated instability is independent of RecA. Note that there is no difference in the level of instability with any of the four constructs in AB1157 versus JC10287. DSB repair, when specifically within the repeat tract, causes significantly increased instability in both strains. All error bars represent +/−2 SEM derived from triplicate experiments.

    Journal: Nucleic Acids Research

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence

    doi: 10.1093/nar/gkm1066

    Figure Lengend Snippet: DSB repair mediated instability of the (GAA•TTC) n sequence is independent of RecA. ( A ) Repair of linear templates obtained by XbaI digestion of GAA-70X, TTC-70X, GAA-30X and TTC-30X resulted in similar levels of instability when transformed into E. coli MM28 [wild-type (WT)] and its isogenic recA mutant strain, M152. There was no difference in the level of repeat instability between the RecA-proficient and RecA-deficient strains, regardless of repeat length or the orientation with respect to the origin of replication. ( B ) Instability of GAA-30X constructs either uncut or following repair at the PstI, XbaI or KpnI restriction sites in isogenic E. coli strains AB1157 (RecA-proficient) and JC10287 (RecA-deficient) showing that DSB repair-mediated instability is independent of RecA. Note that there is no difference in the level of instability with any of the four constructs in AB1157 versus JC10287. DSB repair, when specifically within the repeat tract, causes significantly increased instability in both strains. All error bars represent +/−2 SEM derived from triplicate experiments.

    Article Snippet: The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

    Techniques: Sequencing, Transformation Assay, Mutagenesis, Construct, Derivative Assay

    DSB repair at the center of the (GAA•TTC) n sequence results in the preferential deletion of approximately half (or less than half) of the total repeat length. The residual tract lengths of the (GAA•TTC) n sequence are shown (as a percentage of full-length) after transformation in E. coli MM28 of ( A ) EcoRI and HindIII linearized GAA-79 and TTC-79; ( B ) XbaI linearized GAA-70X and TTC-70X; ( C ) XbaI linearized GAA-30X and TTC-30X, ( D ) XbaI linearized (3′ recessed) and after end filling (blunt-ended) GAA-70X, ( E ) XbaI linearized GAA-30X in MM28 (RecA-proficient) and M152 (RecA-deficient) isogenic strains, and ( F ) XbaI linearized GAA-30X in AB1157 (RecA-proficient) and JC10287 (RecA-deficient) isogenic strains. Note that the size distribution of deletion products is random when DSB repair occurs outside the repeat (EcoRI, HindIII); however, approximately half of the repeat tract, or less, is preferentially deleted when DSB repair occurs at the center of the repeat tract. This is irrespective of the initial length of the repeat tract (70 or 30 triplets), the nature of the termini being repaired (staggered or blunt), or the presence/absence of RecA.

    Journal: Nucleic Acids Research

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence

    doi: 10.1093/nar/gkm1066

    Figure Lengend Snippet: DSB repair at the center of the (GAA•TTC) n sequence results in the preferential deletion of approximately half (or less than half) of the total repeat length. The residual tract lengths of the (GAA•TTC) n sequence are shown (as a percentage of full-length) after transformation in E. coli MM28 of ( A ) EcoRI and HindIII linearized GAA-79 and TTC-79; ( B ) XbaI linearized GAA-70X and TTC-70X; ( C ) XbaI linearized GAA-30X and TTC-30X, ( D ) XbaI linearized (3′ recessed) and after end filling (blunt-ended) GAA-70X, ( E ) XbaI linearized GAA-30X in MM28 (RecA-proficient) and M152 (RecA-deficient) isogenic strains, and ( F ) XbaI linearized GAA-30X in AB1157 (RecA-proficient) and JC10287 (RecA-deficient) isogenic strains. Note that the size distribution of deletion products is random when DSB repair occurs outside the repeat (EcoRI, HindIII); however, approximately half of the repeat tract, or less, is preferentially deleted when DSB repair occurs at the center of the repeat tract. This is irrespective of the initial length of the repeat tract (70 or 30 triplets), the nature of the termini being repaired (staggered or blunt), or the presence/absence of RecA.

    Article Snippet: The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

    Techniques: Sequencing, Transformation Assay

    DSB repair results in dramatically increased instability of the (GAA•TTC) n sequence when the break is located within the repeat tract. ( A ) Representative agarose gels with products of colony PCR showing transformants of GAA-79 [circular (uncut), or linearized with HindIII or EcoRI], and GAA-70X [circular (uncut), or linearized with XbaI]. Arrowheads indicate the position of the full-length repeat tract. DSB repair outside the repeat tract showed levels of instability that were similar to the uncut plasmids; however, DSB repair within the repeat tract resulted in a very high frequency of deletions. The first lane in each gel contains the 1 kb Plus ladder (Invitrogen) with bands from the bottom of the gel representing 0.2, 0.3, 0.4, 0.5 and 0.65 kb (note: the full-length products of GAA-79 and GAA-70X are 423 and 312 bp, respectively, due to the presence of some flanking intron 1 sequence from the human FXN gene in the former). ( B ) Representative agarose gels with products of colony PCR showing transformants of GAA-30X [circular (uncut), or linearized with PstI, XbaI or KpnI]. Arrowheads indicate the position of the full-length repeat tract. DSB repair outside the repeat tract showed levels of instability that were similar to the uncut plasmids; however, DSB repair within the repeat tract resulted in a very high frequency of deletions. Note that even the (GAA•TTC) 30 sequence, which was otherwise extremely stable, showed a dramatic rise in the frequency of deletions. The first lane of every gel contains a DNA size marker; the markers used in the gels containing repair products of templates GAA-30X PstI and KpnI are different from all other gels. The first lane in each gel contains either the 1 kb Plus ladder (Invitrogen) (GAA-30X; PstI and KpnI with bands from the bottom of the gel representing 0.1, 0.2 and 0.3 kb, or the 50 bp DNA ladder (Invitrogen) (GAA-30X; uncut and XbaI) with bands from the bottom of the gel representing 0.1, 0.15, 0.2, 0.25, 0.3 and 0.35 kb. ( C ) DSB repair within a slightly unstable (GAA•TTC) 70–79 sequence produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable in the GAA and TTC orientations. ( D ) DSB repair within a highly stable (GAA•TTC) 30 sequence produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable in the GAA and TTC orientations. All error bars represent +/−2 SEM derived from triplicate experiments.

    Journal: Nucleic Acids Research

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence

    doi: 10.1093/nar/gkm1066

    Figure Lengend Snippet: DSB repair results in dramatically increased instability of the (GAA•TTC) n sequence when the break is located within the repeat tract. ( A ) Representative agarose gels with products of colony PCR showing transformants of GAA-79 [circular (uncut), or linearized with HindIII or EcoRI], and GAA-70X [circular (uncut), or linearized with XbaI]. Arrowheads indicate the position of the full-length repeat tract. DSB repair outside the repeat tract showed levels of instability that were similar to the uncut plasmids; however, DSB repair within the repeat tract resulted in a very high frequency of deletions. The first lane in each gel contains the 1 kb Plus ladder (Invitrogen) with bands from the bottom of the gel representing 0.2, 0.3, 0.4, 0.5 and 0.65 kb (note: the full-length products of GAA-79 and GAA-70X are 423 and 312 bp, respectively, due to the presence of some flanking intron 1 sequence from the human FXN gene in the former). ( B ) Representative agarose gels with products of colony PCR showing transformants of GAA-30X [circular (uncut), or linearized with PstI, XbaI or KpnI]. Arrowheads indicate the position of the full-length repeat tract. DSB repair outside the repeat tract showed levels of instability that were similar to the uncut plasmids; however, DSB repair within the repeat tract resulted in a very high frequency of deletions. Note that even the (GAA•TTC) 30 sequence, which was otherwise extremely stable, showed a dramatic rise in the frequency of deletions. The first lane of every gel contains a DNA size marker; the markers used in the gels containing repair products of templates GAA-30X PstI and KpnI are different from all other gels. The first lane in each gel contains either the 1 kb Plus ladder (Invitrogen) (GAA-30X; PstI and KpnI with bands from the bottom of the gel representing 0.1, 0.2 and 0.3 kb, or the 50 bp DNA ladder (Invitrogen) (GAA-30X; uncut and XbaI) with bands from the bottom of the gel representing 0.1, 0.15, 0.2, 0.25, 0.3 and 0.35 kb. ( C ) DSB repair within a slightly unstable (GAA•TTC) 70–79 sequence produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable in the GAA and TTC orientations. ( D ) DSB repair within a highly stable (GAA•TTC) 30 sequence produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable in the GAA and TTC orientations. All error bars represent +/−2 SEM derived from triplicate experiments.

    Article Snippet: The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

    Techniques: Sequencing, Polymerase Chain Reaction, Marker, Produced, Derivative Assay

    DSB repair-mediated instability of the (GAA•TTC) n sequence is independent of the length of the intervening sequence at the center of the repeat tract. ( A ) GAA-70-spacer construct containing a 28 bp spacer in the XbaI site of GAA-70X, such that BamHI would cut in the center of the spacer (indicated by the black box). ( B ) DSB repair at the BamHI site in GAA-70-spacer construct produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable when transformed into E. coli MM28 [wild-type (WT)] and its isogenic recA mutant strain, M152. All error bars represent +/−2 SEM derived from triplicate experiments. ( C ) DSB repair at the center of the (GAA•TTC) n sequence results in the preferential deletion of approximately half of the total repeat length. The residual tract lengths of the (GAA•TTC) n sequence are shown (as a percentage of full-length) after transformation of BamHI-linearized GAA-70-spacer vector in E. coli MM28 (WT) and M152 ( recA ).

    Journal: Nucleic Acids Research

    Article Title: Repair of DNA double-strand breaks within the (GAAoTTC)n sequence results in frequent deletion of the triplet-repeat sequence

    doi: 10.1093/nar/gkm1066

    Figure Lengend Snippet: DSB repair-mediated instability of the (GAA•TTC) n sequence is independent of the length of the intervening sequence at the center of the repeat tract. ( A ) GAA-70-spacer construct containing a 28 bp spacer in the XbaI site of GAA-70X, such that BamHI would cut in the center of the spacer (indicated by the black box). ( B ) DSB repair at the BamHI site in GAA-70-spacer construct produced a dramatic rise in instability. Note that the (GAA•TTC) n sequence was equally unstable when transformed into E. coli MM28 [wild-type (WT)] and its isogenic recA mutant strain, M152. All error bars represent +/−2 SEM derived from triplicate experiments. ( C ) DSB repair at the center of the (GAA•TTC) n sequence results in the preferential deletion of approximately half of the total repeat length. The residual tract lengths of the (GAA•TTC) n sequence are shown (as a percentage of full-length) after transformation of BamHI-linearized GAA-70-spacer vector in E. coli MM28 (WT) and M152 ( recA ).

    Article Snippet: The GAA-70X construct, which contains the (GAA•TTC)70 sequence with an XbaI site located exactly at the center of the repeat tract , was created using four synthetic oligonucleotides, as follows: Oligo #1: 5′-GGCGCTCCGCTGCAGCC(GAA)35 TCTAGACGCATCGCC-3′ and Oligo #2: 5′GGCGATGCGTCTAGA(TTC)35 GGCTGCAGCGGAGCGCC-3′ were annealed together in 10 mM Tris, pH 8.0, and digestion buffer 3 (100 mM NaCl, 10 mM MgCl2 ) (New England Biolabs), followed by incubating with PstI and XbaI restriction enzymes to digest both ends of the annealed oligos.

    Techniques: Sequencing, Construct, Produced, Transformation Assay, Mutagenesis, Derivative Assay, Plasmid Preparation

    Dendrogram showing the cluster analysis of 76 S. Typhi isolates from Kolkata, India, 2009–2013, by Xba I-PFGE. Band comparison was performed by using the Dice coefficient with 1.5% optimization (Opt) and 1.5% position tolerance (Tol). Pan-susceptible, susceptible to all 17 drugs tested; A, ampicillin; Ac, amoxicillin/clavulanic acid; C, chloramphenicol; Q, co-trimoxazole; T, tetracycline; S, streptomycin; Na, nalidixic acid; Ci, ciprofloxacin; Of, ofloxacin; Le, levofloxacin.

    Journal: PLoS ONE

    Article Title: Antimicrobial Resistance, Virulence Profiles and Molecular Subtypes of Salmonella enterica Serovars Typhi and Paratyphi A Blood Isolates from Kolkata, India during 2009-2013

    doi: 10.1371/journal.pone.0101347

    Figure Lengend Snippet: Dendrogram showing the cluster analysis of 76 S. Typhi isolates from Kolkata, India, 2009–2013, by Xba I-PFGE. Band comparison was performed by using the Dice coefficient with 1.5% optimization (Opt) and 1.5% position tolerance (Tol). Pan-susceptible, susceptible to all 17 drugs tested; A, ampicillin; Ac, amoxicillin/clavulanic acid; C, chloramphenicol; Q, co-trimoxazole; T, tetracycline; S, streptomycin; Na, nalidixic acid; Ci, ciprofloxacin; Of, ofloxacin; Le, levofloxacin.

    Article Snippet: The DNA plugs were digested with 40 U of Xba I (New England Biolabs, MA) at 37°C for 18 h. The digested DNA was run on 1% pulsed field certified agarose gel (Bio-Rad, Hercules, Calif.) prepared in 0.5x TBE buffer (Sigma) using CHEF DRIII (Bio-Rad) apparatus with an initial switch time of 2.2 sec and a final switch time of 63.8 sec at 6 V/cm for 24 h. The gel was stained with ethidium bromide (1 µg/ml, Sigma), destained with deionized water and PFGE profiles were observed with the UV trans-illuminator using GelDoc (Bio-Rad).

    Techniques:

    Dendrogram showing the cluster analysis of 24 S. Paratyphi A isolates from Kolkata, India, 2009–2013, by Xba I-PFGE. Band comparison was performed by using the Dice coefficient with 1.5% optimization (Opt) and 1.5% position tolerance (Tol). Pan-susceptible, susceptible to all 17 drugs tested; Na, nalidixic acid; Ci, ciprofloxacin; Of, ofloxacin; Az, azithromycin.

    Journal: PLoS ONE

    Article Title: Antimicrobial Resistance, Virulence Profiles and Molecular Subtypes of Salmonella enterica Serovars Typhi and Paratyphi A Blood Isolates from Kolkata, India during 2009-2013

    doi: 10.1371/journal.pone.0101347

    Figure Lengend Snippet: Dendrogram showing the cluster analysis of 24 S. Paratyphi A isolates from Kolkata, India, 2009–2013, by Xba I-PFGE. Band comparison was performed by using the Dice coefficient with 1.5% optimization (Opt) and 1.5% position tolerance (Tol). Pan-susceptible, susceptible to all 17 drugs tested; Na, nalidixic acid; Ci, ciprofloxacin; Of, ofloxacin; Az, azithromycin.

    Article Snippet: The DNA plugs were digested with 40 U of Xba I (New England Biolabs, MA) at 37°C for 18 h. The digested DNA was run on 1% pulsed field certified agarose gel (Bio-Rad, Hercules, Calif.) prepared in 0.5x TBE buffer (Sigma) using CHEF DRIII (Bio-Rad) apparatus with an initial switch time of 2.2 sec and a final switch time of 63.8 sec at 6 V/cm for 24 h. The gel was stained with ethidium bromide (1 µg/ml, Sigma), destained with deionized water and PFGE profiles were observed with the UV trans-illuminator using GelDoc (Bio-Rad).

    Techniques:

    (A) PFGE analysis of S . Typhimurium DT104 using XbaI and AvrII. Isolate groups were color identified for simplicity as follows, and the color codes were maintained in all of the assays: reference strain (white); group A, environmental (green); group B,

    Journal: Applied and Environmental Microbiology

    Article Title: Evidence of Metabolic Switching and Implications for Food Safety from the Phenome(s) of Salmonella enterica Serovar Typhimurium DT104 Cultured at Selected Points across the Pork Production Food Chain

    doi: 10.1128/AEM.01041-13

    Figure Lengend Snippet: (A) PFGE analysis of S . Typhimurium DT104 using XbaI and AvrII. Isolate groups were color identified for simplicity as follows, and the color codes were maintained in all of the assays: reference strain (white); group A, environmental (green); group B,

    Article Snippet: Pulsed-field gel electrophoresis (PFGE) was performed using the restriction endonucleases XbaI and AvrII (New England BioLabs) by following the CDC PulseNet protocol ( ).

    Techniques:

    Phosphorylated AlgR and AlgR D54E bound to the fimU promoter. The effect of in vitro phosphorylation of AlgR on binding to an 82-bp fimU DNA fragment was compared with the binding of purified AlgR D54N or AlgR D54E using EMSA. (A) Diagram of the 82-bp fragment used in EMSAs and its location within the fimT-fimU intergenic region. (B) Increasing amounts of the phosphate donor, AcP, were added to the AlgR protein in order to determine the effect of phosphorylation on DNA binding. Wild-type AlgR protein was pretreated with AcP for 30 min prior to binding reactions. Lanes 1 to 8, fimU DNA alone (lane 1) or in the presence of AlgR without AcP (lane 2) or with increasing concentrations of AcP (lanes 3 to 8). Lanes 9 to 12, the AlgR-P–DNA complex was competed with the addition of nonradiolabeled fimU DNA. (C) Phosphorylated AlgR protein was compared with untreated AlgR for DNA binding. Purified AlgR protein was pretreated either with water only or with AcP (500× molar excess of protein) prior to binding reactions. Lanes 1 to 10, fimU DNA incubated with a no-protein control (lane 1), increasing concentrations of AlgR (lanes 2 to 5) or AlgR-P (lanes 6 to 9), or 5 mM AcP only (lane 10). Lanes 11 to 13, 5 nM pscEF DNA incubated with a no-protein control (lane 11), 2.5 μM AlgR (lane 12), or 2.5 μM AlgR-P (lane 13). (D) Purified AlgR D54N or AlgR D54E protein was tested for binding to fimU DNA. Lane 1, no-protein control; lanes 2 to 5, increasing concentrations of AlgR D54N; lanes 6 to 9, increasing concentrations of AlgR D54E (lanes 6 to 9). Open arrowheads, unbound DNA; filled arrowheads, AlgR-DNA complexes. All concentrations given in the figure are final binding reaction concentrations.

    Journal: Journal of Bacteriology

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

    doi: 10.1128/JB.00726-13

    Figure Lengend Snippet: Phosphorylated AlgR and AlgR D54E bound to the fimU promoter. The effect of in vitro phosphorylation of AlgR on binding to an 82-bp fimU DNA fragment was compared with the binding of purified AlgR D54N or AlgR D54E using EMSA. (A) Diagram of the 82-bp fragment used in EMSAs and its location within the fimT-fimU intergenic region. (B) Increasing amounts of the phosphate donor, AcP, were added to the AlgR protein in order to determine the effect of phosphorylation on DNA binding. Wild-type AlgR protein was pretreated with AcP for 30 min prior to binding reactions. Lanes 1 to 8, fimU DNA alone (lane 1) or in the presence of AlgR without AcP (lane 2) or with increasing concentrations of AcP (lanes 3 to 8). Lanes 9 to 12, the AlgR-P–DNA complex was competed with the addition of nonradiolabeled fimU DNA. (C) Phosphorylated AlgR protein was compared with untreated AlgR for DNA binding. Purified AlgR protein was pretreated either with water only or with AcP (500× molar excess of protein) prior to binding reactions. Lanes 1 to 10, fimU DNA incubated with a no-protein control (lane 1), increasing concentrations of AlgR (lanes 2 to 5) or AlgR-P (lanes 6 to 9), or 5 mM AcP only (lane 10). Lanes 11 to 13, 5 nM pscEF DNA incubated with a no-protein control (lane 11), 2.5 μM AlgR (lane 12), or 2.5 μM AlgR-P (lane 13). (D) Purified AlgR D54N or AlgR D54E protein was tested for binding to fimU DNA. Lane 1, no-protein control; lanes 2 to 5, increasing concentrations of AlgR D54N; lanes 6 to 9, increasing concentrations of AlgR D54E (lanes 6 to 9). Open arrowheads, unbound DNA; filled arrowheads, AlgR-DNA complexes. All concentrations given in the figure are final binding reaction concentrations.

    Article Snippet: To create pHERD30T- algR D54E, the codon encoding Asp 54 (GAT) in pHERD30T- algR was mutated to Glu (GAA) using primers algR-D54E-XbaI-F and algR-D54E-XbaI-R with a Phusion site-directed mutagenesis kit (New England BioLabs).

    Techniques: In Vitro, Binding Assay, Purification, Incubation

    AlgR phosphorylation was required for maximal rhamnolipid production. (A) Thin-layer chromatography. Strains PAO1 (WT), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAO1 Δ rhlA (Δ rhlA ) were grown for 16 h in TSB plus 1% glycerol. Mono, monorhamnolipid band; Di, dirhamnolipid band. (B) Strains for which results are shown in panel A were tested for rhamnolipid production by use of the orcinol colorimetric assay and comparison with a standard curve (see Materials and Methods). Values were normalized to total-protein values. (C) The extent of hemolysis due to rhamnolipid production was measured. Cultures were grown for 24 h in a phosphate-limiting medium (tryptic soy broth supplemented with 1% glucose), and cell-free supernatants were heat treated to denature polypeptide-based hemolysins. Serial dilutions were applied to 1% horse red blood cells for 1 h at 37°C, and the percentage of hemolysis was calculated based on total and no hemolysis. Statistical analysis was performed by one-way analysis of variance. n.s., not significant. Asterisks indicate P values of 0.05 to 0.01 (*), 0.01 to 0.001 (**), or

    Journal: Journal of Bacteriology

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

    doi: 10.1128/JB.00726-13

    Figure Lengend Snippet: AlgR phosphorylation was required for maximal rhamnolipid production. (A) Thin-layer chromatography. Strains PAO1 (WT), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAO1 Δ rhlA (Δ rhlA ) were grown for 16 h in TSB plus 1% glycerol. Mono, monorhamnolipid band; Di, dirhamnolipid band. (B) Strains for which results are shown in panel A were tested for rhamnolipid production by use of the orcinol colorimetric assay and comparison with a standard curve (see Materials and Methods). Values were normalized to total-protein values. (C) The extent of hemolysis due to rhamnolipid production was measured. Cultures were grown for 24 h in a phosphate-limiting medium (tryptic soy broth supplemented with 1% glucose), and cell-free supernatants were heat treated to denature polypeptide-based hemolysins. Serial dilutions were applied to 1% horse red blood cells for 1 h at 37°C, and the percentage of hemolysis was calculated based on total and no hemolysis. Statistical analysis was performed by one-way analysis of variance. n.s., not significant. Asterisks indicate P values of 0.05 to 0.01 (*), 0.01 to 0.001 (**), or

    Article Snippet: To create pHERD30T- algR D54E, the codon encoding Asp 54 (GAT) in pHERD30T- algR was mutated to Glu (GAA) using primers algR-D54E-XbaI-F and algR-D54E-XbaI-R with a Phusion site-directed mutagenesis kit (New England BioLabs).

    Techniques: Thin Layer Chromatography, Colorimetric Assay

    Effects of different algR alleles on twitching and swarming motility. (A) Subsurface twitching motilities were analyzed at 48 h after inoculation in LB–1% agar, and the average diameters of twitching zones were measured. Strains PAO1 ( algR + ) (WT), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAZ algR D54E (Δ algZ algR D54 E) were analyzed. PAO1A ( pilA :: Tc ) was used as a negative control. Statistical analysis was performed by one-way analysis of variance. n.s., not significant; ***, P

    Journal: Journal of Bacteriology

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

    doi: 10.1128/JB.00726-13

    Figure Lengend Snippet: Effects of different algR alleles on twitching and swarming motility. (A) Subsurface twitching motilities were analyzed at 48 h after inoculation in LB–1% agar, and the average diameters of twitching zones were measured. Strains PAO1 ( algR + ) (WT), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAZ algR D54E (Δ algZ algR D54 E) were analyzed. PAO1A ( pilA :: Tc ) was used as a negative control. Statistical analysis was performed by one-way analysis of variance. n.s., not significant; ***, P

    Article Snippet: To create pHERD30T- algR D54E, the codon encoding Asp 54 (GAT) in pHERD30T- algR was mutated to Glu (GAA) using primers algR-D54E-XbaI-F and algR-D54E-XbaI-R with a Phusion site-directed mutagenesis kit (New England BioLabs).

    Techniques: Negative Control

    AlgR D54E increased fimU transcription. (A) Diagram showing the genotypes of the following strains: PAO1 ( algR + ), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAZ algR D54E (Δ algZ algR D54E). The dashed regions represent the deletion of the gene indicated. (B) The strains diagramed in panel A were tested for fimU transcriptional activity. Strains contained a fimU :: lacZ transcriptional reporter in the chromosomal attB locus. β-Galactosidase activities from 21-h LB cultures grown at 37°C were assayed. Significant differences from the wild type by Student t tests are indicated (***, P

    Journal: Journal of Bacteriology

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

    doi: 10.1128/JB.00726-13

    Figure Lengend Snippet: AlgR D54E increased fimU transcription. (A) Diagram showing the genotypes of the following strains: PAO1 ( algR + ), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAZ algR D54E (Δ algZ algR D54E). The dashed regions represent the deletion of the gene indicated. (B) The strains diagramed in panel A were tested for fimU transcriptional activity. Strains contained a fimU :: lacZ transcriptional reporter in the chromosomal attB locus. β-Galactosidase activities from 21-h LB cultures grown at 37°C were assayed. Significant differences from the wild type by Student t tests are indicated (***, P

    Article Snippet: To create pHERD30T- algR D54E, the codon encoding Asp 54 (GAT) in pHERD30T- algR was mutated to Glu (GAA) using primers algR-D54E-XbaI-F and algR-D54E-XbaI-R with a Phusion site-directed mutagenesis kit (New England BioLabs).

    Techniques: Activity Assay