ecorv  (New England Biolabs)


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    EcoRV
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    EcoRV 20 000 units
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    r0195l
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    20 000 units
    Category:
    Restriction Enzymes
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    New England Biolabs ecorv
    EcoRV
    EcoRV 20 000 units
    https://www.bioz.com/result/ecorv/product/New England Biolabs
    Average 99 stars, based on 373 article reviews
    Price from $9.99 to $1999.99
    ecorv - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Integrative and Sequence Characteristics of a Novel Genetic Element, ICE6013, in Staphylococcus aureus "

    Article Title: Integrative and Sequence Characteristics of a Novel Genetic Element, ICE6013, in Staphylococcus aureus

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00352-09

    Southern blotting of EcoRV-digested genomic DNA using an ICE 6013 probe. Lanes 1 and 11, DIG-labeled HindIII-digested lambda ladder (Roche); lanes 2 and 10, blank; lanes 3 to 9, seven strains. The arrow indicates the expected 9.6-kb fragment from strain HDG2.
    Figure Legend Snippet: Southern blotting of EcoRV-digested genomic DNA using an ICE 6013 probe. Lanes 1 and 11, DIG-labeled HindIII-digested lambda ladder (Roche); lanes 2 and 10, blank; lanes 3 to 9, seven strains. The arrow indicates the expected 9.6-kb fragment from strain HDG2.

    Techniques Used: Southern Blot, Labeling

    2) Product Images from "Regulated Expression of the Beta2-Toxin Gene (cpb2) in Clostridium perfringens Type A Isolates from Horses with Gastrointestinal Diseases"

    Article Title: Regulated Expression of the Beta2-Toxin Gene (cpb2) in Clostridium perfringens Type A Isolates from Horses with Gastrointestinal Diseases

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.43.8.4002-4009.2005

    RFLP-Southern blot analysis of HpaI- or EcoRV-digested DNA from horse GI disease isolates. Total DNA isolated from each of the specified C. perfringens strains was digested with HpaI (A) or EcoRV (B) and then Southern transferred. The Southern blots were
    Figure Legend Snippet: RFLP-Southern blot analysis of HpaI- or EcoRV-digested DNA from horse GI disease isolates. Total DNA isolated from each of the specified C. perfringens strains was digested with HpaI (A) or EcoRV (B) and then Southern transferred. The Southern blots were

    Techniques Used: Southern Blot, Isolation

    3) Product Images from "Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs)"

    Article Title: Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs)

    Journal: Journal of Nanobiotechnology

    doi: 10.1186/1477-3155-9-7

    Controlled release of USPIO micelles by environmental triggers . (A) Self-assembly of EcoRV-sensitive ssDNA-USPIO clusters, and subsequent enzymatic treatment results in measurable changes in R 2 relaxation coefficient relative to initial values. Following EcoRV treatment, R 2 values return to baseline, a phenomenon that is in significant contrast to the effects of EcoRI treatment of the same clusters (n = 6). * p
    Figure Legend Snippet: Controlled release of USPIO micelles by environmental triggers . (A) Self-assembly of EcoRV-sensitive ssDNA-USPIO clusters, and subsequent enzymatic treatment results in measurable changes in R 2 relaxation coefficient relative to initial values. Following EcoRV treatment, R 2 values return to baseline, a phenomenon that is in significant contrast to the effects of EcoRI treatment of the same clusters (n = 6). * p

    Techniques Used:

    4) Product Images from "Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase"

    Article Title: Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase

    Journal: BMC Microbiology

    doi: 10.1186/s12866-017-1141-0

    Confirmation of the mutation in PA14Δ hepP . PA14 and PA14Δ hepP were grown in LB broth and the chromosomal DNA was extracted. a PCR analysis to detect the presence of MAR2xT7 within hepP . PCR reactions were run using the chromosomal DNA from each strain as a template and primers corresponding to the DNA sequences 94 bp upstream and 179 bp downstream of the hepP structural gene ( zbdP- For3/ hepP- Rev3, Table 2 ). The expected 1926-bp fragment from PA14 (lane 1) and the 2920-bp fragment (the additional 994 bp from MAR2xT7 ) from PA14Δ hepP (lane 2) were detected. Lane 3 is a no-template control and the molecular size standards are in lane 4. b Restriction analysis of the PCR products. The coding sequence for hepP does not contain an EcoR V restriction enzyme site, while MAR2xT7 contains a single EcoR V site. Digestion of the PCR products with EcoR V failed to reduce the size of the 1926-bp fragment obtained from PA14 (lane 3) but resulted in the cleavage of the product obtained from PA14Δ hepP into the expected 800 bp and 2120 bp fragments (lane 4). Lane 1 contains the molecular size standards; lane 2 was left empty
    Figure Legend Snippet: Confirmation of the mutation in PA14Δ hepP . PA14 and PA14Δ hepP were grown in LB broth and the chromosomal DNA was extracted. a PCR analysis to detect the presence of MAR2xT7 within hepP . PCR reactions were run using the chromosomal DNA from each strain as a template and primers corresponding to the DNA sequences 94 bp upstream and 179 bp downstream of the hepP structural gene ( zbdP- For3/ hepP- Rev3, Table 2 ). The expected 1926-bp fragment from PA14 (lane 1) and the 2920-bp fragment (the additional 994 bp from MAR2xT7 ) from PA14Δ hepP (lane 2) were detected. Lane 3 is a no-template control and the molecular size standards are in lane 4. b Restriction analysis of the PCR products. The coding sequence for hepP does not contain an EcoR V restriction enzyme site, while MAR2xT7 contains a single EcoR V site. Digestion of the PCR products with EcoR V failed to reduce the size of the 1926-bp fragment obtained from PA14 (lane 3) but resulted in the cleavage of the product obtained from PA14Δ hepP into the expected 800 bp and 2120 bp fragments (lane 4). Lane 1 contains the molecular size standards; lane 2 was left empty

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Sequencing

    5) Product Images from "Structural diversity of supercoiled DNA"

    Article Title: Structural diversity of supercoiled DNA

    Journal: Nature Communications

    doi: 10.1038/ncomms9440

    Effect of supercoiling on the structure of minicircle DNA. ( a ) Individual 336 bp minicircle topoisomers were isolated and analysed by polyacrylamide gel electrophoresis in the presence of 10 mM CaCl 2 . Mr: 100 bp DNA ladder, L: minicircle linearized by EcoRV, N: minicircle nicked by Nb.BbvCI. ( b ) Projections of cryo-ET subtomograms of hydrated 336 bp DNA minicircles of the Lk =34 topoisomer. ( c ) Commonly observed shapes were open circle, open figure-8, figure-8, racquet, handcuffs, needle, and rod, each of which are shown in orthogonal views. ( d ) Other shapes observed, especially in the more highly supercoiled topoisomers. ( e ) Shape frequency distribution plot for each topoisomer population (n=number of minicircles analysed). A weighted average for each topoisomer, approximating the average degree of compactness, is denoted by the black triangle. The weighted average was calculated by assigning each conformation a value that increased in line with compactness. Open circles were given a value of 1, open figure-8 s a value of 2, figure-8 s as a value of 3, and so on. The relative fraction of each was subsequently used to determine the average degree of compactness. Lk , Δ Lk and superhelical density (σ) for each topoisomer are shown (see Supplementary Note 1 ).
    Figure Legend Snippet: Effect of supercoiling on the structure of minicircle DNA. ( a ) Individual 336 bp minicircle topoisomers were isolated and analysed by polyacrylamide gel electrophoresis in the presence of 10 mM CaCl 2 . Mr: 100 bp DNA ladder, L: minicircle linearized by EcoRV, N: minicircle nicked by Nb.BbvCI. ( b ) Projections of cryo-ET subtomograms of hydrated 336 bp DNA minicircles of the Lk =34 topoisomer. ( c ) Commonly observed shapes were open circle, open figure-8, figure-8, racquet, handcuffs, needle, and rod, each of which are shown in orthogonal views. ( d ) Other shapes observed, especially in the more highly supercoiled topoisomers. ( e ) Shape frequency distribution plot for each topoisomer population (n=number of minicircles analysed). A weighted average for each topoisomer, approximating the average degree of compactness, is denoted by the black triangle. The weighted average was calculated by assigning each conformation a value that increased in line with compactness. Open circles were given a value of 1, open figure-8 s a value of 2, figure-8 s as a value of 3, and so on. The relative fraction of each was subsequently used to determine the average degree of compactness. Lk , Δ Lk and superhelical density (σ) for each topoisomer are shown (see Supplementary Note 1 ).

    Techniques Used: Isolation, Polyacrylamide Gel Electrophoresis

    6) Product Images from "Overexpression of Arabidopsis thaliana gibberellic acid 20 oxidase (AtGA20ox) gene enhance the vegetative growth and fiber quality in kenaf (Hibiscus cannabinus L.) plants"

    Article Title: Overexpression of Arabidopsis thaliana gibberellic acid 20 oxidase (AtGA20ox) gene enhance the vegetative growth and fiber quality in kenaf (Hibiscus cannabinus L.) plants

    Journal: Breeding Science

    doi: 10.1270/jsbbs.65.177

    Selection of putative CaMV35S : AtGA20ox transgenic kenaf plants in MS media containing hygromycin B and the confirmation through PCR amplification and Southern blot analysis. Growth of UT plants in the MS medium without hygromycin B (a) and with hygromycin B (45 μg mL −1 ) (b). Growing of putative transformants in the MS media with hygromycin B (45 μg mL −1 ) (c and d). Detection of the transgene encoding AtGA20ox under duplicate 35S promoter in transgenic G4 and V36 kenaf and UT plants either amplified by PCR using GA-R and 35S-F primers (e and f) or southern blot analysis (g and h). Lane M is ladder, lanes 1–3, 5, 7 (e) and 1–3, 5, 13 (f) are putative transformed G4 and V36 plants respectively showing two amplifications 1.3 kb and 1.6 kb, except for line G4-1 (lane 1 in e) which produced only 1.3 kb amplification product; lane UT is the DNA of untransformed plants; lane −Ve is negative control used PCR product without DNA; lane +Ve is the plasmid DNA of expression clone pEXP32-AtGA20ox. Southern blot analysis was performed with genomic DNA (6 μg–30 μg) digested with EcoR V, separated on 0.8% agarose gel using biotin labelled ORF of AtGA20ox as probe: lane M is marker; lanes 1–3, 5, 7 are putative G4 transformants; lane UT is the DNA of G4 UT and lane +Ve is plasmid DNA (g). In Figure h, lanes 1, 3, 13 are the putative V36 transformants, lane UT is the DNA from V36 UT plant and lane +Ve is plasmid DNA of pEXP32-AtGA20ox digested with EcoR V.
    Figure Legend Snippet: Selection of putative CaMV35S : AtGA20ox transgenic kenaf plants in MS media containing hygromycin B and the confirmation through PCR amplification and Southern blot analysis. Growth of UT plants in the MS medium without hygromycin B (a) and with hygromycin B (45 μg mL −1 ) (b). Growing of putative transformants in the MS media with hygromycin B (45 μg mL −1 ) (c and d). Detection of the transgene encoding AtGA20ox under duplicate 35S promoter in transgenic G4 and V36 kenaf and UT plants either amplified by PCR using GA-R and 35S-F primers (e and f) or southern blot analysis (g and h). Lane M is ladder, lanes 1–3, 5, 7 (e) and 1–3, 5, 13 (f) are putative transformed G4 and V36 plants respectively showing two amplifications 1.3 kb and 1.6 kb, except for line G4-1 (lane 1 in e) which produced only 1.3 kb amplification product; lane UT is the DNA of untransformed plants; lane −Ve is negative control used PCR product without DNA; lane +Ve is the plasmid DNA of expression clone pEXP32-AtGA20ox. Southern blot analysis was performed with genomic DNA (6 μg–30 μg) digested with EcoR V, separated on 0.8% agarose gel using biotin labelled ORF of AtGA20ox as probe: lane M is marker; lanes 1–3, 5, 7 are putative G4 transformants; lane UT is the DNA of G4 UT and lane +Ve is plasmid DNA (g). In Figure h, lanes 1, 3, 13 are the putative V36 transformants, lane UT is the DNA from V36 UT plant and lane +Ve is plasmid DNA of pEXP32-AtGA20ox digested with EcoR V.

    Techniques Used: Selection, Transgenic Assay, Mass Spectrometry, Polymerase Chain Reaction, Amplification, Southern Blot, Transformation Assay, Produced, Negative Control, Plasmid Preparation, Expressing, Agarose Gel Electrophoresis, Marker

    7) Product Images from "Mouse Fyn induces pseudopodium formation in Chinese hamster ovary cells"

    Article Title: Mouse Fyn induces pseudopodium formation in Chinese hamster ovary cells

    Journal: Journal of Veterinary Science

    doi: 10.4142/jvs.2014.15.1.111

    Identification of the recombinant plasmid. (A) Detection of the housekeeping gene GAPDH. (B) Identification of the target gene by RT-PCR. (C) PCR product of the Fyn gene was subcloned into pMD18-T-Fyn and pEGFP-N1-Fyn. (D) Identification of pMD18-T-Fyn fragments produced by restriction enzyme digestion with Eco R I and Sma I. (E) Identification of pEGFP-N1-Fyn fragments generated by restriction enzyme digestion with Eco R I and Sma I. Lane 1, RT-PCR GAPDH product; Lane 2, negative control; Lane 3, RT-PCR Fyn product from brain; Lane 4, positive control; Lane 5, PCR product of pMD18-T-Fyn; Lane 6, PCR product of pEGFP-N1-Fyn; Lanes 7 and 9, positive control; Lanes 8 and 10, recombinant plasmid identification by digestion with EcoR I and Sma I (pMD18-T-Fyn, 2700 bp; pEGFP-N1, 4700 bp; Fyn, 1611 bp); Lane M, DNA marker.
    Figure Legend Snippet: Identification of the recombinant plasmid. (A) Detection of the housekeeping gene GAPDH. (B) Identification of the target gene by RT-PCR. (C) PCR product of the Fyn gene was subcloned into pMD18-T-Fyn and pEGFP-N1-Fyn. (D) Identification of pMD18-T-Fyn fragments produced by restriction enzyme digestion with Eco R I and Sma I. (E) Identification of pEGFP-N1-Fyn fragments generated by restriction enzyme digestion with Eco R I and Sma I. Lane 1, RT-PCR GAPDH product; Lane 2, negative control; Lane 3, RT-PCR Fyn product from brain; Lane 4, positive control; Lane 5, PCR product of pMD18-T-Fyn; Lane 6, PCR product of pEGFP-N1-Fyn; Lanes 7 and 9, positive control; Lanes 8 and 10, recombinant plasmid identification by digestion with EcoR I and Sma I (pMD18-T-Fyn, 2700 bp; pEGFP-N1, 4700 bp; Fyn, 1611 bp); Lane M, DNA marker.

    Techniques Used: Recombinant, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Produced, Generated, Negative Control, Positive Control, Marker

    8) Product Images from "A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting"

    Article Title: A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni049

    ( A ) The components of the pGRFP plasmid vector. The main components are the pUC origin of replication, ampicillin resistance marker and a fused GFP-DsRed gene separated by a linker. The linker region is shown with six amino acid linkers (SGSGSG and GSGSGS) on either side, M13 forward and reverse priming sites, and EcoRV, NotI and SalI sites. ( B ) Flow-cytometry configuration for dual-fluorescence quantification and sorting. A 488 nm laser excites the fluorescent proteins in individual E.coli suspended in the flow stream. The flow cytometer is configured to trigger either on forward scatter or GFP fluorescence. The fluorescence is split using a 550 nm dichroic long pass beam splitter. The green fluorescence is filtered through a 560 nm short pass filter before detection. The red fluorescence passes through a 590 nm long pass filter before detection.
    Figure Legend Snippet: ( A ) The components of the pGRFP plasmid vector. The main components are the pUC origin of replication, ampicillin resistance marker and a fused GFP-DsRed gene separated by a linker. The linker region is shown with six amino acid linkers (SGSGSG and GSGSGS) on either side, M13 forward and reverse priming sites, and EcoRV, NotI and SalI sites. ( B ) Flow-cytometry configuration for dual-fluorescence quantification and sorting. A 488 nm laser excites the fluorescent proteins in individual E.coli suspended in the flow stream. The flow cytometer is configured to trigger either on forward scatter or GFP fluorescence. The fluorescence is split using a 550 nm dichroic long pass beam splitter. The green fluorescence is filtered through a 560 nm short pass filter before detection. The red fluorescence passes through a 590 nm long pass filter before detection.

    Techniques Used: Plasmid Preparation, Marker, Flow Cytometry, Cytometry, Fluorescence

    9) Product Images from "Reconstruction of a replication-competent ancestral murine endogenous retrovirus-L"

    Article Title: Reconstruction of a replication-competent ancestral murine endogenous retrovirus-L

    Journal: Retrovirology

    doi: 10.1186/s12977-018-0416-3

    Integration of ancML into CHO cell DNA. a Example of a genome walker experiment to determine the 3′ flanking sequence of ancML integration events in 15 single cell clones that became resistant to G418 following transfection with ancML. Nested PCR reactions were done using EcoRV digested, adapter ligated, gDNA from single G418-resistat cell clones. Forward and reverse primers were designed to anneal to the R region of the 3′LTR and to the adaptor sequence, respectively. M: molecular weight ladder. u: CHO DNA without an integrated ancML insertion. b Top: the sequence of an integration site with both 5′ and 3′ flanking CHO gDNA. The five-nucleotide target site duplication is indicated in yellow. Bottom: Sequences of 26 ancML integration sites in the CHO genome. Sequences of the ancML U5-PBS region as well as the Leucine (TAA) tRNA sequence are included at the bottom of the diagram. Sequence from the U5 region of the 3′ ancML LTR is indicated in blue. The 5nt linker sequence is indicated in black. The PBS sequence is indicated in purple. CHO genomic sequences are indicted in bold. Dotted lines indicate correspondence of each sequenced 3′ integration junction to the integration site at the top. c Enumeration of G418 resistant colonies of CHO cells transfected with plasmids expressing a neo gene (NEO), L1.3, ancML and an ancML construct with a deletion of the 5nt linker sequence between the 5′ LTR and the PBS (ancML ∆GAAGT). Data are mean ± SD from 3 independent experiments. d Distribution of ancML integration sites in genic, intergenic, or repeat regions relative to matched random controls. The measured value indicates the percentage of ancML integration sites in each population divided by that of the matched random controls (each integration site was matched to three random genomic sequences equidistant to the EcoRV site where the adaptor was ligated). The horizontal dashed line indicates no difference between the frequencies of ancML integration sites in each population compared to the matched controls
    Figure Legend Snippet: Integration of ancML into CHO cell DNA. a Example of a genome walker experiment to determine the 3′ flanking sequence of ancML integration events in 15 single cell clones that became resistant to G418 following transfection with ancML. Nested PCR reactions were done using EcoRV digested, adapter ligated, gDNA from single G418-resistat cell clones. Forward and reverse primers were designed to anneal to the R region of the 3′LTR and to the adaptor sequence, respectively. M: molecular weight ladder. u: CHO DNA without an integrated ancML insertion. b Top: the sequence of an integration site with both 5′ and 3′ flanking CHO gDNA. The five-nucleotide target site duplication is indicated in yellow. Bottom: Sequences of 26 ancML integration sites in the CHO genome. Sequences of the ancML U5-PBS region as well as the Leucine (TAA) tRNA sequence are included at the bottom of the diagram. Sequence from the U5 region of the 3′ ancML LTR is indicated in blue. The 5nt linker sequence is indicated in black. The PBS sequence is indicated in purple. CHO genomic sequences are indicted in bold. Dotted lines indicate correspondence of each sequenced 3′ integration junction to the integration site at the top. c Enumeration of G418 resistant colonies of CHO cells transfected with plasmids expressing a neo gene (NEO), L1.3, ancML and an ancML construct with a deletion of the 5nt linker sequence between the 5′ LTR and the PBS (ancML ∆GAAGT). Data are mean ± SD from 3 independent experiments. d Distribution of ancML integration sites in genic, intergenic, or repeat regions relative to matched random controls. The measured value indicates the percentage of ancML integration sites in each population divided by that of the matched random controls (each integration site was matched to three random genomic sequences equidistant to the EcoRV site where the adaptor was ligated). The horizontal dashed line indicates no difference between the frequencies of ancML integration sites in each population compared to the matched controls

    Techniques Used: Sequencing, Clone Assay, Transfection, Nested PCR, Molecular Weight, Genomic Sequencing, Expressing, Construct

    10) Product Images from "Mec1p associates with functionally compromised telomeres"

    Article Title: Mec1p associates with functionally compromised telomeres

    Journal: Chromosoma

    doi: 10.1007/s00412-011-0359-0

    Mec1-HAp associates with telomeres as cells escape the cell cycle arrest that occurs due to telomere shortening. a Telomere shortening in Mec1-HAp cells lacking the telomerase catalytic subunit Est2p. Cells lacking telomerase were generated by deleting the genomic copy of EST2 , covering the deletion with a plasmid bearing the EST2 gene, and then isolating cells that had lost the plasmid. Cells lacking telomerase were then cultured for the number of population doublings (PD) shown, where the 0 PD sample contains cells that retain the EST2 plasmid and the other PD samples contain cells that lack telomerase. Genomic DNA from each PD was digested with EcoR V and analyzed by Southern blotting using the VI -R PCR product from the primers shown in panel b as probe. Telomeres gradually shortened with increasing PD, allowing the isolation of cells with different telomere lengths. The black arrow designates a background band that hybridizes to probe. The white arrowheads designate rearranged telomeres in PD 70 that are characteristic of the survivor cells that escape the short telomere cell cycle arrest. b A schematic of the loci and primers used to monitor telomere association. The telomeres from the right arm of chromosome VI ( VI -R) and two internal loci from separate chromosomes ( GAL10 and ARO1 ) were PCR amplified in the ChIP experiments in this study. c Analysis of the Mec1-HAp immunoprecipitates using the PCR primers indicated in panel b . Telomere enrichment values are calculated as the ratio of the intensities of the VI -R telomere band to the control ARO1 chromosomal band as described in the “ Materials and methods .” The values shown are averages of multiple determinations. The input dilution is the input DNA that was used to determine the linear range of PCR amplification (see “ Materials and methods ”). d Comparison of cell doubling times and Mec1-HAp telomere enrichments. Mec1-HAp association increases at PD 70 just as survivors are beginning to arise in the culture, as indicated by the simultaneous decrease in doubling time and appearance of rearranged telomeres (panel a )
    Figure Legend Snippet: Mec1-HAp associates with telomeres as cells escape the cell cycle arrest that occurs due to telomere shortening. a Telomere shortening in Mec1-HAp cells lacking the telomerase catalytic subunit Est2p. Cells lacking telomerase were generated by deleting the genomic copy of EST2 , covering the deletion with a plasmid bearing the EST2 gene, and then isolating cells that had lost the plasmid. Cells lacking telomerase were then cultured for the number of population doublings (PD) shown, where the 0 PD sample contains cells that retain the EST2 plasmid and the other PD samples contain cells that lack telomerase. Genomic DNA from each PD was digested with EcoR V and analyzed by Southern blotting using the VI -R PCR product from the primers shown in panel b as probe. Telomeres gradually shortened with increasing PD, allowing the isolation of cells with different telomere lengths. The black arrow designates a background band that hybridizes to probe. The white arrowheads designate rearranged telomeres in PD 70 that are characteristic of the survivor cells that escape the short telomere cell cycle arrest. b A schematic of the loci and primers used to monitor telomere association. The telomeres from the right arm of chromosome VI ( VI -R) and two internal loci from separate chromosomes ( GAL10 and ARO1 ) were PCR amplified in the ChIP experiments in this study. c Analysis of the Mec1-HAp immunoprecipitates using the PCR primers indicated in panel b . Telomere enrichment values are calculated as the ratio of the intensities of the VI -R telomere band to the control ARO1 chromosomal band as described in the “ Materials and methods .” The values shown are averages of multiple determinations. The input dilution is the input DNA that was used to determine the linear range of PCR amplification (see “ Materials and methods ”). d Comparison of cell doubling times and Mec1-HAp telomere enrichments. Mec1-HAp association increases at PD 70 just as survivors are beginning to arise in the culture, as indicated by the simultaneous decrease in doubling time and appearance of rearranged telomeres (panel a )

    Techniques Used: Generated, Plasmid Preparation, Cell Culture, Southern Blot, Polymerase Chain Reaction, Isolation, Amplification, Chromatin Immunoprecipitation

    11) Product Images from "Development of a highly sensitive method for detection of FLT3D835Y"

    Article Title: Development of a highly sensitive method for detection of FLT3D835Y

    Journal: Biomarker Research

    doi: 10.1186/s40364-020-00210-7

    Validation of the RFN-AS-PCR method by using mixtures of DNA samples from FLT3D835Y mutant and wild type AML blood samples. a Screening of 13 AML blood samples by using RFN-AS-PCR identified AML11 as a FLT3D835Y-positive case. b Verification of FLT3D835Y positivity in AML11 by Sanger sequencing. c and d Blood cell DNAs from AML11 and a patient with wild type FLT3 were mixed in the indicated proportions. AS-PCR was performed without EcoRV digestion (panel c ) or with EcoRV digestion (panel d ). Note that the EcoRV digestion increased the detection sensitivity from 1 to 0.001%
    Figure Legend Snippet: Validation of the RFN-AS-PCR method by using mixtures of DNA samples from FLT3D835Y mutant and wild type AML blood samples. a Screening of 13 AML blood samples by using RFN-AS-PCR identified AML11 as a FLT3D835Y-positive case. b Verification of FLT3D835Y positivity in AML11 by Sanger sequencing. c and d Blood cell DNAs from AML11 and a patient with wild type FLT3 were mixed in the indicated proportions. AS-PCR was performed without EcoRV digestion (panel c ) or with EcoRV digestion (panel d ). Note that the EcoRV digestion increased the detection sensitivity from 1 to 0.001%

    Techniques Used: Polymerase Chain Reaction, Mutagenesis, Sequencing

    Development of RFN-AS-PCR for detection of FLT3D835Y. a . Schematic illustration of the RFN-AS-PCR method. b and c The sensitivities of nested AS-PCR and RFN-AS-PCR methods were determined by using purified plasmid DNAs. Mixtures of FLT3 plasmid DNAs containing the indicated percentages of the FLT3D835Y mutant were amplified with primers F1_5 and F1_3. The PCR products were left undigested ( b ) or digested with restriction enzyme EcoRV ( c ) and then subjected to nested AS-PCR analyses with a primer mixture containing F1_5n, F1_3n, F1_mut, and F1_wt. The final PCR products were analyzed on 3% agarose gel, and DNA bands were visualized by staining with SYBR green. The positions of wild type FLT3- and mutant FLT3D835Y-specfic products are indicated. The higher molecular weight bands shared by both wild type FLT3 and mutant FLT3D835Y are products of primer pairs F1_5n/F1_3n (403 bp), F1_5/F1_3, F1_5n/F1_3, and/or F1_5/F1_3n
    Figure Legend Snippet: Development of RFN-AS-PCR for detection of FLT3D835Y. a . Schematic illustration of the RFN-AS-PCR method. b and c The sensitivities of nested AS-PCR and RFN-AS-PCR methods were determined by using purified plasmid DNAs. Mixtures of FLT3 plasmid DNAs containing the indicated percentages of the FLT3D835Y mutant were amplified with primers F1_5 and F1_3. The PCR products were left undigested ( b ) or digested with restriction enzyme EcoRV ( c ) and then subjected to nested AS-PCR analyses with a primer mixture containing F1_5n, F1_3n, F1_mut, and F1_wt. The final PCR products were analyzed on 3% agarose gel, and DNA bands were visualized by staining with SYBR green. The positions of wild type FLT3- and mutant FLT3D835Y-specfic products are indicated. The higher molecular weight bands shared by both wild type FLT3 and mutant FLT3D835Y are products of primer pairs F1_5n/F1_3n (403 bp), F1_5/F1_3, F1_5n/F1_3, and/or F1_5/F1_3n

    Techniques Used: Polymerase Chain Reaction, Purification, Plasmid Preparation, Mutagenesis, Amplification, Agarose Gel Electrophoresis, Staining, SYBR Green Assay, Molecular Weight

    12) Product Images from "CRISPR/Cas9 Targets Chicken Embryonic Somatic Cells In Vitro and In Vivo and generates Phenotypic Abnormalities"

    Article Title: CRISPR/Cas9 Targets Chicken Embryonic Somatic Cells In Vitro and In Vivo and generates Phenotypic Abnormalities

    Journal: Scientific Reports

    doi: 10.1038/srep34524

    In vitro analysis of NHEJ and HDR genome modification (arrows) mediated by sgRNA-Cas9 system in chicken cell lines. ( a ) Frequency (%) of NHEJ mutation mediated by KIAA1279, Cdkn1b and Mbd3 -targeting sgRNA-Cas9 system in chicken DF-1 cells by PCR and T7E1 assay. 1kM- 1 kbp DNA ladder, M- 100 bp DNA ladder. ( b ) Frequency (%) of NHEJ mutation mediated by KIAA1279 and Cdkn1b -targeting sgRNA-CRISPR/Cas9 system in chicken lymphoma B DT40 cells by PCR and T7E1 assay. ( c ) Representative gel from DF-1 cells transfected with the RET-targeting sgRNA-Cas9 and the ssODN showing efficient integration of the HDR-based BamHI and EcoRV sequence. The frequency of HDR is represented in percentages. 1-No sgRNA, 2- MEN2B sgRNA #1 plus ssODN, 3- MEN2B sgRNA #1 and #2 plus ssODN and 4- MEN2A/HSCR sgRNA 1 plus ssODN. ( d ) Representative gel for single cell clones derived from DF-1 cells transfected with the RET-targeting sgRNA-Cas9 and the ssODN for the MEN2B and MEN2A/HSCR HDR modifications respectively. The table shows the ratio of the monoallelic and biallelic HDR-based mutations detected with single cell clones and the overall efficiency in percentage: N = 19 for MEN2B and N = 12 for MEN2A/HSCR.
    Figure Legend Snippet: In vitro analysis of NHEJ and HDR genome modification (arrows) mediated by sgRNA-Cas9 system in chicken cell lines. ( a ) Frequency (%) of NHEJ mutation mediated by KIAA1279, Cdkn1b and Mbd3 -targeting sgRNA-Cas9 system in chicken DF-1 cells by PCR and T7E1 assay. 1kM- 1 kbp DNA ladder, M- 100 bp DNA ladder. ( b ) Frequency (%) of NHEJ mutation mediated by KIAA1279 and Cdkn1b -targeting sgRNA-CRISPR/Cas9 system in chicken lymphoma B DT40 cells by PCR and T7E1 assay. ( c ) Representative gel from DF-1 cells transfected with the RET-targeting sgRNA-Cas9 and the ssODN showing efficient integration of the HDR-based BamHI and EcoRV sequence. The frequency of HDR is represented in percentages. 1-No sgRNA, 2- MEN2B sgRNA #1 plus ssODN, 3- MEN2B sgRNA #1 and #2 plus ssODN and 4- MEN2A/HSCR sgRNA 1 plus ssODN. ( d ) Representative gel for single cell clones derived from DF-1 cells transfected with the RET-targeting sgRNA-Cas9 and the ssODN for the MEN2B and MEN2A/HSCR HDR modifications respectively. The table shows the ratio of the monoallelic and biallelic HDR-based mutations detected with single cell clones and the overall efficiency in percentage: N = 19 for MEN2B and N = 12 for MEN2A/HSCR.

    Techniques Used: In Vitro, Non-Homologous End Joining, Modification, Mutagenesis, Polymerase Chain Reaction, CRISPR, Transfection, Sequencing, Clone Assay, Derivative Assay

    13) Product Images from "Significance of the Bacteriophage Treatment Schedule in Reducing Salmonella Colonization of Poultry"

    Article Title: Significance of the Bacteriophage Treatment Schedule in Reducing Salmonella Colonization of Poultry

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01257-12

    DNA restriction patterns of the bacteriophages UAB_Phi20 (lanes 3 to 5), UAB_Phi87 (lanes 6 to 8), and UAB_Phi78 (lanes 9 to 11) with the restriction enzymes EcoRI (lanes 3, 6, and 9), EcoRV (lanes 4, 7, and 10), and HindIII (lanes 5, 8, and 11). Lanes
    Figure Legend Snippet: DNA restriction patterns of the bacteriophages UAB_Phi20 (lanes 3 to 5), UAB_Phi87 (lanes 6 to 8), and UAB_Phi78 (lanes 9 to 11) with the restriction enzymes EcoRI (lanes 3, 6, and 9), EcoRV (lanes 4, 7, and 10), and HindIII (lanes 5, 8, and 11). Lanes

    Techniques Used:

    14) Product Images from "Reconstruction of a replication-competent ancestral murine endogenous retrovirus-L"

    Article Title: Reconstruction of a replication-competent ancestral murine endogenous retrovirus-L

    Journal: Retrovirology

    doi: 10.1186/s12977-018-0416-3

    Integration of ancML into CHO cell DNA. a Example of a genome walker experiment to determine the 3′ flanking sequence of ancML integration events in 15 single cell clones that became resistant to G418 following transfection with ancML. Nested PCR reactions were done using EcoRV digested, adapter ligated, gDNA from single G418-resistat cell clones. Forward and reverse primers were designed to anneal to the R region of the 3′LTR and to the adaptor sequence, respectively. M: molecular weight ladder. u: CHO DNA without an integrated ancML insertion. b Top: the sequence of an integration site with both 5′ and 3′ flanking CHO gDNA. The five-nucleotide target site duplication is indicated in yellow. Bottom: Sequences of 26 ancML integration sites in the CHO genome. Sequences of the ancML U5-PBS region as well as the Leucine (TAA) tRNA sequence are included at the bottom of the diagram. Sequence from the U5 region of the 3′ ancML LTR is indicated in blue. The 5nt linker sequence is indicated in black. The PBS sequence is indicated in purple. CHO genomic sequences are indicted in bold. Dotted lines indicate correspondence of each sequenced 3′ integration junction to the integration site at the top. c Enumeration of G418 resistant colonies of CHO cells transfected with plasmids expressing a neo gene (NEO), L1.3, ancML and an ancML construct with a deletion of the 5nt linker sequence between the 5′ LTR and the PBS (ancML ∆GAAGT). Data are mean ± SD from 3 independent experiments. d Distribution of ancML integration sites in genic, intergenic, or repeat regions relative to matched random controls. The measured value indicates the percentage of ancML integration sites in each population divided by that of the matched random controls (each integration site was matched to three random genomic sequences equidistant to the EcoRV site where the adaptor was ligated). The horizontal dashed line indicates no difference between the frequencies of ancML integration sites in each population compared to the matched controls
    Figure Legend Snippet: Integration of ancML into CHO cell DNA. a Example of a genome walker experiment to determine the 3′ flanking sequence of ancML integration events in 15 single cell clones that became resistant to G418 following transfection with ancML. Nested PCR reactions were done using EcoRV digested, adapter ligated, gDNA from single G418-resistat cell clones. Forward and reverse primers were designed to anneal to the R region of the 3′LTR and to the adaptor sequence, respectively. M: molecular weight ladder. u: CHO DNA without an integrated ancML insertion. b Top: the sequence of an integration site with both 5′ and 3′ flanking CHO gDNA. The five-nucleotide target site duplication is indicated in yellow. Bottom: Sequences of 26 ancML integration sites in the CHO genome. Sequences of the ancML U5-PBS region as well as the Leucine (TAA) tRNA sequence are included at the bottom of the diagram. Sequence from the U5 region of the 3′ ancML LTR is indicated in blue. The 5nt linker sequence is indicated in black. The PBS sequence is indicated in purple. CHO genomic sequences are indicted in bold. Dotted lines indicate correspondence of each sequenced 3′ integration junction to the integration site at the top. c Enumeration of G418 resistant colonies of CHO cells transfected with plasmids expressing a neo gene (NEO), L1.3, ancML and an ancML construct with a deletion of the 5nt linker sequence between the 5′ LTR and the PBS (ancML ∆GAAGT). Data are mean ± SD from 3 independent experiments. d Distribution of ancML integration sites in genic, intergenic, or repeat regions relative to matched random controls. The measured value indicates the percentage of ancML integration sites in each population divided by that of the matched random controls (each integration site was matched to three random genomic sequences equidistant to the EcoRV site where the adaptor was ligated). The horizontal dashed line indicates no difference between the frequencies of ancML integration sites in each population compared to the matched controls

    Techniques Used: Sequencing, Clone Assay, Transfection, Nested PCR, Molecular Weight, Genomic Sequencing, Expressing, Construct

    15) Product Images from "Flexible and scalable genotyping-by-sequencing strategies for population studies"

    Article Title: Flexible and scalable genotyping-by-sequencing strategies for population studies

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-15-979

    Effect of read count on marker dataset size and imputation. Fraction of shared post-filter, pre-imputation genetic markers and fraction of post-imputation shared genome with original sample for subsamplings of A) RsaI F 2 -44 and B) HincII F 2 -23. C) Imputed genomes for each subsample in RsaI F 2 -44 displayed in concentric rings. Sample read count declines from the outermost ring to the innermost. D) Imputed genomes for each subsample in HincII F 2 -23 displayed in concentric circles. Sample read count declines from the outermost ring to the innermost.
    Figure Legend Snippet: Effect of read count on marker dataset size and imputation. Fraction of shared post-filter, pre-imputation genetic markers and fraction of post-imputation shared genome with original sample for subsamplings of A) RsaI F 2 -44 and B) HincII F 2 -23. C) Imputed genomes for each subsample in RsaI F 2 -44 displayed in concentric rings. Sample read count declines from the outermost ring to the innermost. D) Imputed genomes for each subsample in HincII F 2 -23 displayed in concentric circles. Sample read count declines from the outermost ring to the innermost.

    Techniques Used: Marker

    Trait mapping for yellowy ( y1 ) and sugary ( su1 ) in an F 2 admixture population. A green line in the plots annotates the locations of both genes. Pre-imputation markers are shown in black and grey. Markers, post-imputation and error correction are shown in color. A) RsaI GBS dataset, su1 map. B) RsaI GBS dataset, y1 map. C) HincII GBS dataset, su1 map. D) RsaI GBS dataset, y1 map.
    Figure Legend Snippet: Trait mapping for yellowy ( y1 ) and sugary ( su1 ) in an F 2 admixture population. A green line in the plots annotates the locations of both genes. Pre-imputation markers are shown in black and grey. Markers, post-imputation and error correction are shown in color. A) RsaI GBS dataset, su1 map. B) RsaI GBS dataset, y1 map. C) HincII GBS dataset, su1 map. D) RsaI GBS dataset, y1 map.

    Techniques Used:

    Fraction of predicted sites covered in samples from a F 2 admixture population. Reads from each F 2 sample were aligned to predicted sites, then predicted sites were placed in 2 bp bins, with the fraction covered in each bin indicated by the heatmap. A) The RsaI dataset, aligned against total predicted sites. B) RsaI dataset, aligned against the subset of predicted sites with sequencing coverage in the original RsaI B73 GBS experiment. C) HincII dataset, aligned against total predicted sites. D) HincII dataset, aligned against predicted sites with at least one read coverage in the original HincII experiment. Sample order is given, left to right, in Additional file 5 : Table S1.
    Figure Legend Snippet: Fraction of predicted sites covered in samples from a F 2 admixture population. Reads from each F 2 sample were aligned to predicted sites, then predicted sites were placed in 2 bp bins, with the fraction covered in each bin indicated by the heatmap. A) The RsaI dataset, aligned against total predicted sites. B) RsaI dataset, aligned against the subset of predicted sites with sequencing coverage in the original RsaI B73 GBS experiment. C) HincII dataset, aligned against total predicted sites. D) HincII dataset, aligned against predicted sites with at least one read coverage in the original HincII experiment. Sample order is given, left to right, in Additional file 5 : Table S1.

    Techniques Used: Sequencing

    16) Product Images from "Characterization of human adenovirus 35 and derivation of complex vectors"

    Article Title: Characterization of human adenovirus 35 and derivation of complex vectors

    Journal: Virology Journal

    doi: 10.1186/1743-422X-7-276

    Effect of E3 deletions on fiber transcription and virus fitness . (A) Northern blot of 24 hpi total RNA from 293-ORF6 cells infected with wild type Ad35 (wt), E1-deleted rAd35 (d8), or rAd35 d8 constructs with the E3 deletions noted in panel B. The blot was hybridized with a fiber (top) or pIX probe (bottom); the fiber and pIX transcripts are labeled. Numbers on the right denote migration of RNA size standards (kilobases). (B) Schematic of the Ad35 E3 region and E3 deletions (not to scale). The coordinates of the nucleotides that form the deletion junctions are shown above the lines. L4 poly(A), E3 poly(A) = L4 and E3 polyadenylation hexanucleotide signals, respectively. (C) and (D) Viral vector growth competitions. rAd35 d8 E1-deleted vector was mixed with (C) rAd35 d8, E3(X)-deleted vector or with (D) rAd35 d8, E3(HE)-deleted vector. Relative change in genome amounts was determined by DNA restriction fragment analysis of the input mixture of viruses and each serial passage. DNA restriction fragment analysis uniquely identified each virus genome, which is indicated to the left of the gel and their fragment sizes in bp on the right. I = input mixed viruses used for initial infection; M = mock infected cells; P1 = initial infection; P4 = fourth passage; a, b, c = replicates. Restriction enzymes EcoRV and BlpI were used in panels C and D respectively.
    Figure Legend Snippet: Effect of E3 deletions on fiber transcription and virus fitness . (A) Northern blot of 24 hpi total RNA from 293-ORF6 cells infected with wild type Ad35 (wt), E1-deleted rAd35 (d8), or rAd35 d8 constructs with the E3 deletions noted in panel B. The blot was hybridized with a fiber (top) or pIX probe (bottom); the fiber and pIX transcripts are labeled. Numbers on the right denote migration of RNA size standards (kilobases). (B) Schematic of the Ad35 E3 region and E3 deletions (not to scale). The coordinates of the nucleotides that form the deletion junctions are shown above the lines. L4 poly(A), E3 poly(A) = L4 and E3 polyadenylation hexanucleotide signals, respectively. (C) and (D) Viral vector growth competitions. rAd35 d8 E1-deleted vector was mixed with (C) rAd35 d8, E3(X)-deleted vector or with (D) rAd35 d8, E3(HE)-deleted vector. Relative change in genome amounts was determined by DNA restriction fragment analysis of the input mixture of viruses and each serial passage. DNA restriction fragment analysis uniquely identified each virus genome, which is indicated to the left of the gel and their fragment sizes in bp on the right. I = input mixed viruses used for initial infection; M = mock infected cells; P1 = initial infection; P4 = fourth passage; a, b, c = replicates. Restriction enzymes EcoRV and BlpI were used in panels C and D respectively.

    Techniques Used: Northern Blot, Infection, Construct, Labeling, Migration, Plasmid Preparation

    17) Product Images from "CpG Island Methylation in a Mouse Model of Lymphoma Is Driven by the Genetic Configuration of Tumor Cells"

    Article Title: CpG Island Methylation in a Mouse Model of Lymphoma Is Driven by the Genetic Configuration of Tumor Cells

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0030167

    Signature of Aberrant DNA Methylation in MYC -Induced T cell Lymphomas (A) Schematic representation of transgenes in MYC -induced T cell lymphoma model used in this study. (B) Examples of RLGS profiles obtained from normal EμSR-tTA thymocytes (normal) and EμSR-tTA;Teto-MYC tumors (tumor). Loss or decreased intensity of single-copy NotI fragments in the tumors, relative to several neighboring unaltered fragments, was detected by visual inspection of overlaid autoradiographs. The arrows indicate the position of RLGS fragments 4E41, 2E46, 4B22, and 4D09, which are lost or reduced in intensity in tumor samples, indicative of specific methylation events. (C) Summary of the frequency of methylation events observed in eight tumor samples and two normal thymic controls. Filled boxes indicate methylation at that site and open boxes indicate no methylation. Only data for RLGS fragments that have been methylated in more than 50% of tumors are shown. Methylation data from the analysis of three sets of EGFP-positive and EGFP-negative thymocytes isolated from 21-d-old EμSR-tTA;Teto-MYC;Teto-Cre;Rosa26LOXP EGFP mice are also shown (see text and Figure 2 ). (D) Southern blot analysis of control and tumor cells using the 2C51 NotI-EcoRV DNA fragment as a probe. DNA isolated from thymocytes of EμSR-tTA mice was digested either with EcoRV alone (lane 1) or with EcoRV and NotI (lanes 2 and 3). DNA isolated from eight different T cell lymphomas (tumors; lanes 1–8) was digested with restriction enzymes EcoRV and NotI. The bands represent methylated (M) or unmethylated (U) NotI sites. (E) Schematic representation of a CpG island located near the RLGS fragment 3F56. Nucleotide position of the NotI site (black triangle), exon 1, and the region amplified for bisulfite sequencing is indicated relative to the transcription initiation site. Bisulfite sequencing was performed on DNA isolated from two normal and seven tumor samples. Each circle represents a CpG dinucleotide and each row of circles indicates the sequence of an individual allele. Filled black circles correspond to methylated CpG dinucleotides and open circles represent unmethylated CpG dinucleotides. (F) Expression of genes near the RLGS fragments 2C51 and 3F56 was quantified in thymocytes isolated from three EμSR-tTA mice (open bars) and eight EμSR-tTA;Teto-MYC tumors (black bars) using real-time RT-PCR. The y -axis represents fold decrease in gene expression relative to the levels of gene expression detected in the first control.
    Figure Legend Snippet: Signature of Aberrant DNA Methylation in MYC -Induced T cell Lymphomas (A) Schematic representation of transgenes in MYC -induced T cell lymphoma model used in this study. (B) Examples of RLGS profiles obtained from normal EμSR-tTA thymocytes (normal) and EμSR-tTA;Teto-MYC tumors (tumor). Loss or decreased intensity of single-copy NotI fragments in the tumors, relative to several neighboring unaltered fragments, was detected by visual inspection of overlaid autoradiographs. The arrows indicate the position of RLGS fragments 4E41, 2E46, 4B22, and 4D09, which are lost or reduced in intensity in tumor samples, indicative of specific methylation events. (C) Summary of the frequency of methylation events observed in eight tumor samples and two normal thymic controls. Filled boxes indicate methylation at that site and open boxes indicate no methylation. Only data for RLGS fragments that have been methylated in more than 50% of tumors are shown. Methylation data from the analysis of three sets of EGFP-positive and EGFP-negative thymocytes isolated from 21-d-old EμSR-tTA;Teto-MYC;Teto-Cre;Rosa26LOXP EGFP mice are also shown (see text and Figure 2 ). (D) Southern blot analysis of control and tumor cells using the 2C51 NotI-EcoRV DNA fragment as a probe. DNA isolated from thymocytes of EμSR-tTA mice was digested either with EcoRV alone (lane 1) or with EcoRV and NotI (lanes 2 and 3). DNA isolated from eight different T cell lymphomas (tumors; lanes 1–8) was digested with restriction enzymes EcoRV and NotI. The bands represent methylated (M) or unmethylated (U) NotI sites. (E) Schematic representation of a CpG island located near the RLGS fragment 3F56. Nucleotide position of the NotI site (black triangle), exon 1, and the region amplified for bisulfite sequencing is indicated relative to the transcription initiation site. Bisulfite sequencing was performed on DNA isolated from two normal and seven tumor samples. Each circle represents a CpG dinucleotide and each row of circles indicates the sequence of an individual allele. Filled black circles correspond to methylated CpG dinucleotides and open circles represent unmethylated CpG dinucleotides. (F) Expression of genes near the RLGS fragments 2C51 and 3F56 was quantified in thymocytes isolated from three EμSR-tTA mice (open bars) and eight EμSR-tTA;Teto-MYC tumors (black bars) using real-time RT-PCR. The y -axis represents fold decrease in gene expression relative to the levels of gene expression detected in the first control.

    Techniques Used: DNA Methylation Assay, RLGS, Methylation, Isolation, Mouse Assay, Southern Blot, Amplification, Methylation Sequencing, Sequencing, Expressing, Quantitative RT-PCR

    18) Product Images from "The Core Histone N-Terminal Tail Domains Negatively Regulate Binding of Transcription Factor IIIA to a Nucleosome Containing a 5S RNA Gene via a Novel Mechanism"

    Article Title: The Core Histone N-Terminal Tail Domains Negatively Regulate Binding of Transcription Factor IIIA to a Nucleosome Containing a 5S RNA Gene via a Novel Mechanism

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.25.1.241-249.2005

    Spontaneous DNA unwrapping from the 5S nucleosome (nuc) is stimulated two- to fivefold upon removal of the core histone tail domains. Nucleosomes reconstituted with the 215-bp 5S DNA fragment were selected to eliminate minor translational positions, incubated with trypsin agarose beads to remove core histone tail domains, and digested with either EcoRV or BbvI. A 154-bp truncated 5S DNA fragment was included in each digest as a naked DNA reference. (A) Proteins within intact or tailless nucleosomes were examined on SDS-18% PAGE. Lane 1, (H3/H4) 2 ). (C) Intact or tailless 5S nucleosomes and 154-bp DNA fragments were digested with the indicated enzymes, and the logarithm of the uncut fraction was plotted versus digestion times. Note that the nucleosomal curves were generated from digests containing 5,000 or 600 U of EcoRV or BbvI, respectively, while the naked DNA curves were generated from identical reaction mixtures containing 10-U/ml concentrations of each enzyme. Diamonds, intact nucleosome; squares, tailless nucleosome; and triangles, naked DNA. (C) K conf eq . W.t. and tryp, intact and tailless nucleosomes, respectively.
    Figure Legend Snippet: Spontaneous DNA unwrapping from the 5S nucleosome (nuc) is stimulated two- to fivefold upon removal of the core histone tail domains. Nucleosomes reconstituted with the 215-bp 5S DNA fragment were selected to eliminate minor translational positions, incubated with trypsin agarose beads to remove core histone tail domains, and digested with either EcoRV or BbvI. A 154-bp truncated 5S DNA fragment was included in each digest as a naked DNA reference. (A) Proteins within intact or tailless nucleosomes were examined on SDS-18% PAGE. Lane 1, (H3/H4) 2 ). (C) Intact or tailless 5S nucleosomes and 154-bp DNA fragments were digested with the indicated enzymes, and the logarithm of the uncut fraction was plotted versus digestion times. Note that the nucleosomal curves were generated from digests containing 5,000 or 600 U of EcoRV or BbvI, respectively, while the naked DNA curves were generated from identical reaction mixtures containing 10-U/ml concentrations of each enzyme. Diamonds, intact nucleosome; squares, tailless nucleosome; and triangles, naked DNA. (C) K conf eq . W.t. and tryp, intact and tailless nucleosomes, respectively.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, Generated

    19) Product Images from "Evidence that Plasmid-Borne Botulinum Neurotoxin Type B Genes Are Widespread among Clostridium botulinum Serotype B Strains"

    Article Title: Evidence that Plasmid-Borne Botulinum Neurotoxin Type B Genes Are Widespread among Clostridium botulinum Serotype B Strains

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004829

    BamHI, HindIII, SacI and EcoRV restrictions of the bont /B PCR products obtained from strains CDC-1758 (lanes 1, 5, 9, 14); CDC-1828 (lanes 2, 6, 10, 15); CDC-1436 (lanes 3, 7, 11, 16); CDC-4848 (lanes 4, 8, 12); CDC-816 (lane 13). M.S. (Molecular standard, 1 kb Promega).
    Figure Legend Snippet: BamHI, HindIII, SacI and EcoRV restrictions of the bont /B PCR products obtained from strains CDC-1758 (lanes 1, 5, 9, 14); CDC-1828 (lanes 2, 6, 10, 15); CDC-1436 (lanes 3, 7, 11, 16); CDC-4848 (lanes 4, 8, 12); CDC-816 (lane 13). M.S. (Molecular standard, 1 kb Promega).

    Techniques Used: Polymerase Chain Reaction

    20) Product Images from "Interneuron dysfunction in a new knock-in mouse model of SCN1A GEFS+"

    Article Title: Interneuron dysfunction in a new knock-in mouse model of SCN1A GEFS+

    Journal: bioRxiv

    doi: 10.1101/849240

    Generation of Scn1a KT/+ mouse using CRISPR/Cas9 technology. (A) GEFS+ causing K1270T mutation (asterisk) is located in S2 transmembrane segment of domain III of the alpha subunit of Nav1.1 ion channel encoded by the human SCN1A gene. (B) Location of the guide RNA relative to the Cas9 cut site and the locus of the K1259T mutation in the mouse Scn1a gene and the Nav1.1 protein sequence. Repair template sequence with the base pair changes introducing the K-T mutation and the EcoRV cut site. All edited nucleotides are shown in lowercase letters and the homology-dependent repair region is represented by underlined letters. Two additional silent mutations (asterisks) were added to prevent re-cutting by Cas9 following HDR. (C) Outline of the steps followed to generate transgenic K1270T mouse colonies in B6NJ and 129×1 genetic backgrounds via CRISPR/Cas9 gene editing. (D) DNA sequence comparison between a wild-type ( Scn1a +/+ ) and a heterozygous ( Scn1a KT/+ ) mouse showing missense K-T mutation and another silent mutation that results in an EcoRV cut site. (E) A representative agarose gel shows PCR amplified DNA bands digested with EcoRV which distinguishes between mice homozygous for the mutant allele, Scn1a KT/KT (223 bp and 165 bp), Scn1a +/+ wild-type mice homozygous for the wild-type allele (388bp) and heterozygous Scn1a KT/+ mice carrying one copy each of wild-type (388 bp) and mutant (223 and 165 bp) alleles.
    Figure Legend Snippet: Generation of Scn1a KT/+ mouse using CRISPR/Cas9 technology. (A) GEFS+ causing K1270T mutation (asterisk) is located in S2 transmembrane segment of domain III of the alpha subunit of Nav1.1 ion channel encoded by the human SCN1A gene. (B) Location of the guide RNA relative to the Cas9 cut site and the locus of the K1259T mutation in the mouse Scn1a gene and the Nav1.1 protein sequence. Repair template sequence with the base pair changes introducing the K-T mutation and the EcoRV cut site. All edited nucleotides are shown in lowercase letters and the homology-dependent repair region is represented by underlined letters. Two additional silent mutations (asterisks) were added to prevent re-cutting by Cas9 following HDR. (C) Outline of the steps followed to generate transgenic K1270T mouse colonies in B6NJ and 129×1 genetic backgrounds via CRISPR/Cas9 gene editing. (D) DNA sequence comparison between a wild-type ( Scn1a +/+ ) and a heterozygous ( Scn1a KT/+ ) mouse showing missense K-T mutation and another silent mutation that results in an EcoRV cut site. (E) A representative agarose gel shows PCR amplified DNA bands digested with EcoRV which distinguishes between mice homozygous for the mutant allele, Scn1a KT/KT (223 bp and 165 bp), Scn1a +/+ wild-type mice homozygous for the wild-type allele (388bp) and heterozygous Scn1a KT/+ mice carrying one copy each of wild-type (388 bp) and mutant (223 and 165 bp) alleles.

    Techniques Used: CRISPR, Mutagenesis, Sequencing, Transgenic Assay, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Mouse Assay

    21) Product Images from "Longevity and resistance to stress correlate with DNA repair capacity in Caenorhabditis elegans"

    Article Title: Longevity and resistance to stress correlate with DNA repair capacity in Caenorhabditis elegans

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm1161

    Detection of UV-induced pyrimidine dimers in VPS-45. C. elegans genomic DNA was digested with EcoR V, electrophoresed, transferred to membrane by Southern blot, and hybridized to radio-labeled DNA probe. ( A ) Genomic map of the VPS-45 gene. “ EcoR V” indicates location of EcoR V restriction sites outside of VPS-45 . The black boxes indicate exons. The position of the DNA probe used in this study is shown below the gene. ( B ) Southern blot in glyoxal-DMSO buffer. Lanes 1 and 2, 1 μg genomic DNA; lanes 3 and 4, 3 μg genomic DNA. DNA was irradiated with UV light, treated with (+) or without (−) T4 endonuclease, electrophoresed in glyoxal/DMSO, and analyzed by Southern blot. Results are shown for EcoR V digested C. elegans genomic DNA ( C ).
    Figure Legend Snippet: Detection of UV-induced pyrimidine dimers in VPS-45. C. elegans genomic DNA was digested with EcoR V, electrophoresed, transferred to membrane by Southern blot, and hybridized to radio-labeled DNA probe. ( A ) Genomic map of the VPS-45 gene. “ EcoR V” indicates location of EcoR V restriction sites outside of VPS-45 . The black boxes indicate exons. The position of the DNA probe used in this study is shown below the gene. ( B ) Southern blot in glyoxal-DMSO buffer. Lanes 1 and 2, 1 μg genomic DNA; lanes 3 and 4, 3 μg genomic DNA. DNA was irradiated with UV light, treated with (+) or without (−) T4 endonuclease, electrophoresed in glyoxal/DMSO, and analyzed by Southern blot. Results are shown for EcoR V digested C. elegans genomic DNA ( C ).

    Techniques Used: Southern Blot, Labeling, Irradiation

    22) Product Images from "Comparison between single and multi-locus approaches for specimen identification in Mytilus mussels"

    Article Title: Comparison between single and multi-locus approaches for specimen identification in Mytilus mussels

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-55855-8

    Restriction map of markers RFLP-PCR ( a ) Me15-16 Aci I, ( b ) ITS Hha I, ( c ) CO I Xba I and ( d ) 16S rRNA EcoR V, Nhe I and Spe I. *Is used to identify the new haplotypes found in this work. For clarity, we will conserve the name M. galloprovincialis to refer the former Northern Hemisphere haplotype and use M. chilensis for the former Southern Hemisphere haplotype.
    Figure Legend Snippet: Restriction map of markers RFLP-PCR ( a ) Me15-16 Aci I, ( b ) ITS Hha I, ( c ) CO I Xba I and ( d ) 16S rRNA EcoR V, Nhe I and Spe I. *Is used to identify the new haplotypes found in this work. For clarity, we will conserve the name M. galloprovincialis to refer the former Northern Hemisphere haplotype and use M. chilensis for the former Southern Hemisphere haplotype.

    Techniques Used: Polymerase Chain Reaction, Northern Blot

    23) Product Images from "Nonviral Gene Targeting at rDNA Locus of Human Mesenchymal Stem Cells"

    Article Title: Nonviral Gene Targeting at rDNA Locus of Human Mesenchymal Stem Cells

    Journal: BioMed Research International

    doi: 10.1155/2013/135189

    Site-specific integration at the rDNA locus of MSCs. (a) Schematic of the construction of pHr2-NL. pHr2-NL contained two inverted expression cassettes, one consisting of an IRES element from the encephalomyocarditis virus, the coding region of the Neo gene, the SV40 polyA signal (SV40pA), and two loxP sites with the same orientation. LoxP sites were recognized by CRE enzyme to remove the Neo cassette after gene targeting. LHA, long homologous arm (U13369:937-6523); SHA, short homologous arm (U13369:6523–7643). The genomic locus indicates the 6.7 kb fragment (U13369:937-7643) required for homologous recombination at the internal transcribed spacer 1 (ITS1) of the rRNA gene. Single cutting sites for restricted enzymes of Nco I, EcoR I, Hind III , and Pvu II are located at the IRES-Neo frame and outside of the long homologous fragment. The fragment between the two Pvu II sites was 8285 bp in size, and it was detected using probe 1 (P1). The expected sizes of the restriction fragments produced by Nco I, EcoR I, and Hind III were 4001 bp, 7628 bp, and 15,316 bp, respectively. These were detected using probe 2 (P2). Primer t-up was located at the SV40 polyA. Primer t-re was located outside of the SHA at the hrDNA locus. (b) Drug-resistant cell in basal medium. (c) Drug-resistant colonies in the medium supplemented with VEGF+bFGF+Vc+ITS-X. (d) Identification of colonies with site-specific integration by PCR. The expected fragment, 1.3 kb in size, was amplified from the genomic DNA of colonies using site-specific integration. M, DL200 DNA marker; 1, negative colony; 2–5, positive colonies; 6, wild-type MSCs. (e–f) Southern blotting analysis of the representative recombinants. Genomic DNA digested with Pvu II , Nco I, EcoR I, and Hind III was analyzed. A specific band was consistently detected in colonies 1-1, 1-2, 2-1, and 2-2. An additional band beside the specific band was detected in colony 2-3. c, control (untransfected MSCs); N, Nco I; E, EcoR I; H, Hind III .
    Figure Legend Snippet: Site-specific integration at the rDNA locus of MSCs. (a) Schematic of the construction of pHr2-NL. pHr2-NL contained two inverted expression cassettes, one consisting of an IRES element from the encephalomyocarditis virus, the coding region of the Neo gene, the SV40 polyA signal (SV40pA), and two loxP sites with the same orientation. LoxP sites were recognized by CRE enzyme to remove the Neo cassette after gene targeting. LHA, long homologous arm (U13369:937-6523); SHA, short homologous arm (U13369:6523–7643). The genomic locus indicates the 6.7 kb fragment (U13369:937-7643) required for homologous recombination at the internal transcribed spacer 1 (ITS1) of the rRNA gene. Single cutting sites for restricted enzymes of Nco I, EcoR I, Hind III , and Pvu II are located at the IRES-Neo frame and outside of the long homologous fragment. The fragment between the two Pvu II sites was 8285 bp in size, and it was detected using probe 1 (P1). The expected sizes of the restriction fragments produced by Nco I, EcoR I, and Hind III were 4001 bp, 7628 bp, and 15,316 bp, respectively. These were detected using probe 2 (P2). Primer t-up was located at the SV40 polyA. Primer t-re was located outside of the SHA at the hrDNA locus. (b) Drug-resistant cell in basal medium. (c) Drug-resistant colonies in the medium supplemented with VEGF+bFGF+Vc+ITS-X. (d) Identification of colonies with site-specific integration by PCR. The expected fragment, 1.3 kb in size, was amplified from the genomic DNA of colonies using site-specific integration. M, DL200 DNA marker; 1, negative colony; 2–5, positive colonies; 6, wild-type MSCs. (e–f) Southern blotting analysis of the representative recombinants. Genomic DNA digested with Pvu II , Nco I, EcoR I, and Hind III was analyzed. A specific band was consistently detected in colonies 1-1, 1-2, 2-1, and 2-2. An additional band beside the specific band was detected in colony 2-3. c, control (untransfected MSCs); N, Nco I; E, EcoR I; H, Hind III .

    Techniques Used: Expressing, Homologous Recombination, Produced, Polymerase Chain Reaction, Amplification, Marker, Southern Blot

    24) Product Images from "Mitotic post-translational modifications of histones promote chromatin compaction in vitro"

    Article Title: Mitotic post-translational modifications of histones promote chromatin compaction in vitro

    Journal: Open Biology

    doi: 10.1098/rsob.170076

    Purified histones can be used to reconstitute chromatin on the 601-197-25 array. ( a ) Scheme of chromatin analysis. After reconstitution the quality of chromatin was assessed by digestion with BsrBI and micrococcal nuclease. Fully assembled chromatin was subjected to analysis by a variety of microscopy methods. ( b ) Vector containing 601-197-25 array. The array is flanked by EcoRV sites. Each 601 monomer contains a BsrBI site. ( c ) BsrBI digestion was carried out on chromatin assembled with an increasing ratio of histones : 601 monomers. Unassembled array was digested into 197 fragments. The presence of a nucleosome on a 601 monomer led to the protection of the BsrBI site, generating longer fragments. When fully saturated the array could no longer be digested. ( d ) Micrococcal nuclease digestion of unreconstituted DNA and chromatin reconstituted with interphase and mitotic derived histones. The presence of an approximately 200 bp ladder confirms the presence of nucleosomes.
    Figure Legend Snippet: Purified histones can be used to reconstitute chromatin on the 601-197-25 array. ( a ) Scheme of chromatin analysis. After reconstitution the quality of chromatin was assessed by digestion with BsrBI and micrococcal nuclease. Fully assembled chromatin was subjected to analysis by a variety of microscopy methods. ( b ) Vector containing 601-197-25 array. The array is flanked by EcoRV sites. Each 601 monomer contains a BsrBI site. ( c ) BsrBI digestion was carried out on chromatin assembled with an increasing ratio of histones : 601 monomers. Unassembled array was digested into 197 fragments. The presence of a nucleosome on a 601 monomer led to the protection of the BsrBI site, generating longer fragments. When fully saturated the array could no longer be digested. ( d ) Micrococcal nuclease digestion of unreconstituted DNA and chromatin reconstituted with interphase and mitotic derived histones. The presence of an approximately 200 bp ladder confirms the presence of nucleosomes.

    Techniques Used: Purification, Microscopy, Plasmid Preparation, Derivative Assay

    25) Product Images from "Specific cleavage of DNA molecules at RecA-mediated triple-strand structure"

    Article Title: Specific cleavage of DNA molecules at RecA-mediated triple-strand structure

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh004

    Determination of the nuclease S1 cleavage site. Linearized pBR322 DNA was subjected to triple-strand cleavage with a deoxyoligonucleotide terminating the 5′ termini with either cytosine (oligo C), adenine (oligo D), thymine (oligo E) or guanine (oligo F). A portion of the DNA from each sample was electrophoresed on agarose gel and another portion of the DNA, after treating with SspI or EcoRV and labeling the 5′ termini with 32 P, was electrophoresed on a sequence gel and autoradiographed. ( A ) Diagrammatic representation of the positions and directions of deoxyoligonucleotides used for triplex cleavage. Nucleotide numbers (nt. numbers) are those registered in GenBank. The distances in the diagram do not necessarily reflect actual distances. ( B ) Agarose gel electrophoresis patterns of the products. Lane 1, products with oligo C; lane 2, products with oligo D; lane 3, products with oligo E; and lane 4, products with oligo F. In lane M, we show molecular size markers. ( C ) Autoradigraphic patterns of DNA after digestion with SspI and terminal labeling. ( D ) Auto radigraphic patterns of DNA after digestion with EcoRV and terminal labeling. (C and D) Lane 1, products with oligo C; lane 2, products with oligo D; lane 3, products with oligo E; and lane 4, products with oligo F. We also show sequence patterns of the control M13mp18 DNA on the left-hand side (lane M) and size of the DNA fragments (nt) on the right-hand side.
    Figure Legend Snippet: Determination of the nuclease S1 cleavage site. Linearized pBR322 DNA was subjected to triple-strand cleavage with a deoxyoligonucleotide terminating the 5′ termini with either cytosine (oligo C), adenine (oligo D), thymine (oligo E) or guanine (oligo F). A portion of the DNA from each sample was electrophoresed on agarose gel and another portion of the DNA, after treating with SspI or EcoRV and labeling the 5′ termini with 32 P, was electrophoresed on a sequence gel and autoradiographed. ( A ) Diagrammatic representation of the positions and directions of deoxyoligonucleotides used for triplex cleavage. Nucleotide numbers (nt. numbers) are those registered in GenBank. The distances in the diagram do not necessarily reflect actual distances. ( B ) Agarose gel electrophoresis patterns of the products. Lane 1, products with oligo C; lane 2, products with oligo D; lane 3, products with oligo E; and lane 4, products with oligo F. In lane M, we show molecular size markers. ( C ) Autoradigraphic patterns of DNA after digestion with SspI and terminal labeling. ( D ) Auto radigraphic patterns of DNA after digestion with EcoRV and terminal labeling. (C and D) Lane 1, products with oligo C; lane 2, products with oligo D; lane 3, products with oligo E; and lane 4, products with oligo F. We also show sequence patterns of the control M13mp18 DNA on the left-hand side (lane M) and size of the DNA fragments (nt) on the right-hand side.

    Techniques Used: Agarose Gel Electrophoresis, Labeling, Sequencing

    26) Product Images from "Dynamic structures of Bacillus subtilis RecN-DNA complexes"

    Article Title: Dynamic structures of Bacillus subtilis RecN-DNA complexes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm759

    RecN binding to nicked DNA. Nt.AlwI-treated EcoRV-linear DNA (150 nM in nt) and RecN (10 nM) were incubated for 10 min at 37°C. Reaction was fixed with 0.1 glutaraldehyde and deposited on freshly cut mica. ( A ) AFM images of Nt.AlwI-treated DNA–RecN complexes analysed. ( B ) Geometrical analyses of Nt.AlwI-treated DNA–RecN complexes. The length from the end of the substrate to DNA-bound RecN was measured and shown as a histogram in 250 bp bin. A schematic map of plasmid DNA with the target localization for Nt.AlwI endonuclease and relevant high dA + dT regions ( P lac , pUC ori and f1 ori ) are shown at the same scale of the histogram.
    Figure Legend Snippet: RecN binding to nicked DNA. Nt.AlwI-treated EcoRV-linear DNA (150 nM in nt) and RecN (10 nM) were incubated for 10 min at 37°C. Reaction was fixed with 0.1 glutaraldehyde and deposited on freshly cut mica. ( A ) AFM images of Nt.AlwI-treated DNA–RecN complexes analysed. ( B ) Geometrical analyses of Nt.AlwI-treated DNA–RecN complexes. The length from the end of the substrate to DNA-bound RecN was measured and shown as a histogram in 250 bp bin. A schematic map of plasmid DNA with the target localization for Nt.AlwI endonuclease and relevant high dA + dT regions ( P lac , pUC ori and f1 ori ) are shown at the same scale of the histogram.

    Techniques Used: Binding Assay, Incubation, Plasmid Preparation

    27) Product Images from "Flexible and scalable genotyping-by-sequencing strategies for population studies"

    Article Title: Flexible and scalable genotyping-by-sequencing strategies for population studies

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-15-979

    Fraction of predicted sites covered in samples from a F 2 admixture population. Reads from each F 2 sample were aligned to predicted sites, then predicted sites were placed in 2 bp bins, with the fraction covered in each bin indicated by the heatmap. A) The RsaI dataset, aligned against total predicted sites. B) RsaI dataset, aligned against the subset of predicted sites with sequencing coverage in the original RsaI B73 GBS experiment. C) HincII dataset, aligned against total predicted sites. D) HincII dataset, aligned against predicted sites with at least one read coverage in the original HincII experiment. Sample order is given, left to right, in Additional file 5 : Table S1.
    Figure Legend Snippet: Fraction of predicted sites covered in samples from a F 2 admixture population. Reads from each F 2 sample were aligned to predicted sites, then predicted sites were placed in 2 bp bins, with the fraction covered in each bin indicated by the heatmap. A) The RsaI dataset, aligned against total predicted sites. B) RsaI dataset, aligned against the subset of predicted sites with sequencing coverage in the original RsaI B73 GBS experiment. C) HincII dataset, aligned against total predicted sites. D) HincII dataset, aligned against predicted sites with at least one read coverage in the original HincII experiment. Sample order is given, left to right, in Additional file 5 : Table S1.

    Techniques Used: Sequencing

    28) Product Images from "Molecular Epidemiology, Sequence Types, and Plasmid Analyses of KPC-Producing Klebsiella pneumoniae Strains in Israel ▿"

    Article Title: Molecular Epidemiology, Sequence Types, and Plasmid Analyses of KPC-Producing Klebsiella pneumoniae Strains in Israel ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01818-09

    A comparison of bla KPC-2 -carrying plasmids originated from two K. pneumoniae clones and two E. coli clones isolated in the same time period. Plasmid restriction analysis of transformants carrying these plasmids showed identity between the K. pneumoniae plasmids (K) and the E. coli plasmids (E). Plasmids from both organisms were digested with BglII (lanes 2 to 5), EcoRV (lanes 6 to 9), and SmaI (lanes 10 to 13) prior to electrophoresis. GeneRuler 1-kb DNA ladder (Fermentas Life Sciences), lane 1 (M); E. coli 386, lanes 2, 6, and 10; K. pneumoniae 523 PFGE type R, lanes 3, 7, and 11; E. coli 547, lanes 4, 8, and 12); K. pneumoniae 531, PFGE type, lanes 5, 9, and 13.
    Figure Legend Snippet: A comparison of bla KPC-2 -carrying plasmids originated from two K. pneumoniae clones and two E. coli clones isolated in the same time period. Plasmid restriction analysis of transformants carrying these plasmids showed identity between the K. pneumoniae plasmids (K) and the E. coli plasmids (E). Plasmids from both organisms were digested with BglII (lanes 2 to 5), EcoRV (lanes 6 to 9), and SmaI (lanes 10 to 13) prior to electrophoresis. GeneRuler 1-kb DNA ladder (Fermentas Life Sciences), lane 1 (M); E. coli 386, lanes 2, 6, and 10; K. pneumoniae 523 PFGE type R, lanes 3, 7, and 11; E. coli 547, lanes 4, 8, and 12); K. pneumoniae 531, PFGE type, lanes 5, 9, and 13.

    Techniques Used: Clone Assay, Isolation, Plasmid Preparation, Electrophoresis

    29) Product Images from "Inferring the annual migration patterns of fall armyworm (Lepidoptera: Noctuidae) in the United States from mitochondrial haplotypes"

    Article Title: Inferring the annual migration patterns of fall armyworm (Lepidoptera: Noctuidae) in the United States from mitochondrial haplotypes

    Journal: Ecology and Evolution

    doi: 10.1002/ece3.268

    Diagram of the portion of mitochondrial Cytochrome Oxidase I ( COI ) gene used to identify strain and the individual corn-strain (CS) haplotypes. The putative translational start site of the COI gene was arbitrarily designated as coordinate 0. Short block arrows indicate location and direction of the COI-893F and COI-1303R primers used for PCR amplification and DNA sequencing. Vertical lines within the COI gene identify polymorphic sites with the rice-strain (RS) specific EcoRV site identified. All polymorphic sites except 1287 were used to identify or confirm strain identity. The CS-h haplotypes were determined by the polymorphisms at site 1164 and 1287.
    Figure Legend Snippet: Diagram of the portion of mitochondrial Cytochrome Oxidase I ( COI ) gene used to identify strain and the individual corn-strain (CS) haplotypes. The putative translational start site of the COI gene was arbitrarily designated as coordinate 0. Short block arrows indicate location and direction of the COI-893F and COI-1303R primers used for PCR amplification and DNA sequencing. Vertical lines within the COI gene identify polymorphic sites with the rice-strain (RS) specific EcoRV site identified. All polymorphic sites except 1287 were used to identify or confirm strain identity. The CS-h haplotypes were determined by the polymorphisms at site 1164 and 1287.

    Techniques Used: Blocking Assay, Polymerase Chain Reaction, Amplification, DNA Sequencing

    30) Product Images from "Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds"

    Article Title: Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-10-183

    Southern blot analysis of GS2 genes in Medicago truncatula . A. Schematic representation of BAC mth2-53e90 indicating the position of MtGS2a (AC148968-43) and MtGS2b (AC 1448968-42) and the restriction sites relevant for the southern analysis. The position of the probe used for the Southern analysis is also indicated. B. Southern hybridization of M. truncatula J5 genomic DNA and BAC mth2-53e90 DNA. 20 μg of genomic DNA and 5 μg of BAC DNA were digested with BgLII (1), NcoI (2) and EcoRV (3) and probed with a 260 bp DNA fragment corresponding to part of the 5'UTR and coding sequence of GS2a cDNA.
    Figure Legend Snippet: Southern blot analysis of GS2 genes in Medicago truncatula . A. Schematic representation of BAC mth2-53e90 indicating the position of MtGS2a (AC148968-43) and MtGS2b (AC 1448968-42) and the restriction sites relevant for the southern analysis. The position of the probe used for the Southern analysis is also indicated. B. Southern hybridization of M. truncatula J5 genomic DNA and BAC mth2-53e90 DNA. 20 μg of genomic DNA and 5 μg of BAC DNA were digested with BgLII (1), NcoI (2) and EcoRV (3) and probed with a 260 bp DNA fragment corresponding to part of the 5'UTR and coding sequence of GS2a cDNA.

    Techniques Used: Southern Blot, BAC Assay, Hybridization, Sequencing

    31) Product Images from "Investigation of DNA-protein Sequence-Specific Interactions with a ds-DNA Array"

    Article Title: Investigation of DNA-protein Sequence-Specific Interactions with a ds-DNA Array

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    doi: 10.3390/10020417

    The images of EcoR I and Rsa I digestion on ds-DNA array. (A): the image of the ds-DNA array which created by insertion with Cy3-dUTP (a) digested by EcoR I under 1h(b) and 12h(c); (B): the image of ds-DNA array (a) digested by Rsa I under 1 h(b) and 12 h(c) digestion. The four different concentrations of Oligo II (C, 1, 2, 3 and 4 below the two images indicate control, 80 µM, 40 µM, 20 µM and 10 µM Oligo II, respectively.) were spotted onto the array.
    Figure Legend Snippet: The images of EcoR I and Rsa I digestion on ds-DNA array. (A): the image of the ds-DNA array which created by insertion with Cy3-dUTP (a) digested by EcoR I under 1h(b) and 12h(c); (B): the image of ds-DNA array (a) digested by Rsa I under 1 h(b) and 12 h(c) digestion. The four different concentrations of Oligo II (C, 1, 2, 3 and 4 below the two images indicate control, 80 µM, 40 µM, 20 µM and 10 µM Oligo II, respectively.) were spotted onto the array.

    Techniques Used: DNA Array

    The fluorescence intensity variation of EcoR I and Rsa I digestion on the same array after methylation. EM indicated methylation enzyme and ER indicated EcoR I.
    Figure Legend Snippet: The fluorescence intensity variation of EcoR I and Rsa I digestion on the same array after methylation. EM indicated methylation enzyme and ER indicated EcoR I.

    Techniques Used: Fluorescence, Methylation

    The images of array one digested by EcoR I and Rsa I after methylation. (A): Images of array one which created by inserted Cy3-dUTP (a) was treated by EcoR I methylation enzyme (b) and then by EcoR I (c); (B): Images of array one which created by inserted Cy3-dUTP (a) was treated by EcoR I methylation enzyme (b) and then by Rsa I (c). (C and E below the images indicate Control probe and Oligo I, respectively)
    Figure Legend Snippet: The images of array one digested by EcoR I and Rsa I after methylation. (A): Images of array one which created by inserted Cy3-dUTP (a) was treated by EcoR I methylation enzyme (b) and then by EcoR I (c); (B): Images of array one which created by inserted Cy3-dUTP (a) was treated by EcoR I methylation enzyme (b) and then by Rsa I (c). (C and E below the images indicate Control probe and Oligo I, respectively)

    Techniques Used: Methylation

    32) Product Images from "Identification of five novel mutations in the long isoform of the USH2A gene in Chinese families with Usher syndrome type II"

    Article Title: Identification of five novel mutations in the long isoform of the USH2A gene in Chinese families with Usher syndrome type II

    Journal: Molecular Vision

    doi:

    Restriction fragment length analysis on the three mutations detected in this study. A : c.99_100insT created a Taq a I restriction site that cosegregated with the affected individuals and the carriers (370 bp, 470 bp, and 840 bp), but not with unaffected individuals and normal controls (840bp). B: c.8483delC created an MslI restriction site that cosegregated with the affected individuals and the carriers (150 bp, 340 bp, and 490 bp), but not with unaffected individuals and normal control (490 bp). C : c.9450G > A created an EcoRV restriction site that cosegregated with the affected individuals and the carriers (265 bp, and 530 bp), but not with unaffected individuals and normal control (530 bp). Participant identification number is given above each lane. N represents the normal control, and M refers to the DNA ladder in bp.
    Figure Legend Snippet: Restriction fragment length analysis on the three mutations detected in this study. A : c.99_100insT created a Taq a I restriction site that cosegregated with the affected individuals and the carriers (370 bp, 470 bp, and 840 bp), but not with unaffected individuals and normal controls (840bp). B: c.8483delC created an MslI restriction site that cosegregated with the affected individuals and the carriers (150 bp, 340 bp, and 490 bp), but not with unaffected individuals and normal control (490 bp). C : c.9450G > A created an EcoRV restriction site that cosegregated with the affected individuals and the carriers (265 bp, and 530 bp), but not with unaffected individuals and normal control (530 bp). Participant identification number is given above each lane. N represents the normal control, and M refers to the DNA ladder in bp.

    Techniques Used:

    33) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    34) Product Images from "Intact nucleosomal context enables chromodomain reader MRG15 to distinguish H3K36me3 from -me2"

    Article Title: Intact nucleosomal context enables chromodomain reader MRG15 to distinguish H3K36me3 from -me2

    Journal: bioRxiv

    doi: 10.1101/2020.04.30.070136

    Pulldown Experiment with GST- or his6-tagged MRG15. A, Nucleosomes, comprising 186 bp biotinylated DNA, were immobilised on streptavidin dynabeads and beads were preincubated with BSA at a blocking step. Unbound nucleosomes and Bovine Serum Albumin (BSA) were then washed from the beads with 250 mM KCl buffer. Subsequently, the beads were incubated with GST-MRG15 or GST alone (negative control) with BSA and salmon sperm DNA to out-compete non-specific interactions. Unbound species were then removed in wash steps, and the beads analysed by SDS-PAGE and Western blot. B, As in A, except using his6-MRG15 in binding buffer containing 250 mM KCl or 150 mM NaCl. Unbound species were removed in wash steps, and the beads analysed by SDS-PAGE and Western blot.
    Figure Legend Snippet: Pulldown Experiment with GST- or his6-tagged MRG15. A, Nucleosomes, comprising 186 bp biotinylated DNA, were immobilised on streptavidin dynabeads and beads were preincubated with BSA at a blocking step. Unbound nucleosomes and Bovine Serum Albumin (BSA) were then washed from the beads with 250 mM KCl buffer. Subsequently, the beads were incubated with GST-MRG15 or GST alone (negative control) with BSA and salmon sperm DNA to out-compete non-specific interactions. Unbound species were then removed in wash steps, and the beads analysed by SDS-PAGE and Western blot. B, As in A, except using his6-MRG15 in binding buffer containing 250 mM KCl or 150 mM NaCl. Unbound species were removed in wash steps, and the beads analysed by SDS-PAGE and Western blot.

    Techniques Used: Blocking Assay, Incubation, Negative Control, SDS Page, Western Blot, Binding Assay

    35) Product Images from "Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo"

    Article Title: Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo

    Journal: RNA

    doi: 10.1261/rna.035097.112

    Validation of fluorescence reporters in vivo. ( A ) Western blots from primary cultures further verify enrichment of epithelial and mesenchymal markers and splicing factors in keratinocytes and fibroblasts, respectively. (K) Keratinocytes; (F) fibroblasts. ( B ) Endogenous FGFR2 transcripts from keratinocyte and fibroblast cultures contain restriction enzyme sites specific to the IIIb and IIIc isoforms, respectively. The fibroblast culture contains a minority of IIIb transcripts, which is likely due to a small amount of keratinocyte contamination observed in the fibroblast cultures. (L) 1 Kb Plus Ladder (Invitrogen); (M) mock-digested RT-PCR amplicon; (A) IIIb-specific restriction digestion with AvaI; (E) IIIc-specific restriction digestion with EcoRV. Positions and sizes of AvaI and EcoRV cleavage products are indicated with arrows to the left and right of the gel, respectively. ( C ) RT-PCR revealed that reporter transcripts from pRint/pGIIIcI 2 keratinocyte primary cultures (K) predominantly skipped IIIc ( bottom band), while transcripts from fibroblast primary cell lines (F) contained IIIc within the reporter ( top band). The cartoon depiction to the right indicates the RT-PCR products of the EGFP transcript (gray boxes) with and without the IIIc exon (white box). ( D ) Despite robust expression of DsRed, almost no EGFP was observed in whole tails, epidermis, dermis, epidermal-derived keratinocytes, or dermal-derived fibroblasts from pRint/pGIIIcI ΔΔ mice. Conversely, tails, epidermis, and primary keratinocytes displayed strong EGFP and DsRed expression, while dermis and dermal-derived primary fibroblasts nearly exclusively produced DsRed and not EGFP. Two contaminating keratinocytes in the pRint/pGIIIcI 2 fibroblast culture are visible in the DsRed channel (white arrow).
    Figure Legend Snippet: Validation of fluorescence reporters in vivo. ( A ) Western blots from primary cultures further verify enrichment of epithelial and mesenchymal markers and splicing factors in keratinocytes and fibroblasts, respectively. (K) Keratinocytes; (F) fibroblasts. ( B ) Endogenous FGFR2 transcripts from keratinocyte and fibroblast cultures contain restriction enzyme sites specific to the IIIb and IIIc isoforms, respectively. The fibroblast culture contains a minority of IIIb transcripts, which is likely due to a small amount of keratinocyte contamination observed in the fibroblast cultures. (L) 1 Kb Plus Ladder (Invitrogen); (M) mock-digested RT-PCR amplicon; (A) IIIb-specific restriction digestion with AvaI; (E) IIIc-specific restriction digestion with EcoRV. Positions and sizes of AvaI and EcoRV cleavage products are indicated with arrows to the left and right of the gel, respectively. ( C ) RT-PCR revealed that reporter transcripts from pRint/pGIIIcI 2 keratinocyte primary cultures (K) predominantly skipped IIIc ( bottom band), while transcripts from fibroblast primary cell lines (F) contained IIIc within the reporter ( top band). The cartoon depiction to the right indicates the RT-PCR products of the EGFP transcript (gray boxes) with and without the IIIc exon (white box). ( D ) Despite robust expression of DsRed, almost no EGFP was observed in whole tails, epidermis, dermis, epidermal-derived keratinocytes, or dermal-derived fibroblasts from pRint/pGIIIcI ΔΔ mice. Conversely, tails, epidermis, and primary keratinocytes displayed strong EGFP and DsRed expression, while dermis and dermal-derived primary fibroblasts nearly exclusively produced DsRed and not EGFP. Two contaminating keratinocytes in the pRint/pGIIIcI 2 fibroblast culture are visible in the DsRed channel (white arrow).

    Techniques Used: Fluorescence, In Vivo, Western Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Expressing, Derivative Assay, Mouse Assay, Produced

    36) Product Images from "Evaluation of a Cocktail of Three Bacteriophages for Biocontrol of Escherichia coli O157:H7"

    Article Title: Evaluation of a Cocktail of Three Bacteriophages for Biocontrol of Escherichia coli O157:H7

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.70.6.3417-3424.2004

    Electron micrographs of phages negatively stained with 1% uranyl acetate; restriction digest of phage DNA restricted with EcoRV and phage plaque morphology. (A) Phage e4/1c and associated plaque. (B) Phage e11/2 and associated plaque. (C) Phage pp01 and associated plaque. Bar, 100 nm. Lanes: 1, 1 kb; 2, T4 DNA; 3, e4/1c DNA; 4, e11/2 DNA; 5, pp01 DNA. The DNA was run on a 0.7% agarose gel.
    Figure Legend Snippet: Electron micrographs of phages negatively stained with 1% uranyl acetate; restriction digest of phage DNA restricted with EcoRV and phage plaque morphology. (A) Phage e4/1c and associated plaque. (B) Phage e11/2 and associated plaque. (C) Phage pp01 and associated plaque. Bar, 100 nm. Lanes: 1, 1 kb; 2, T4 DNA; 3, e4/1c DNA; 4, e11/2 DNA; 5, pp01 DNA. The DNA was run on a 0.7% agarose gel.

    Techniques Used: Staining, Agarose Gel Electrophoresis

    37) Product Images from "Restriction endonucleases that cleave RNA/DNA heteroduplexes bind dsDNA in A-like conformation"

    Article Title: Restriction endonucleases that cleave RNA/DNA heteroduplexes bind dsDNA in A-like conformation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa403

    Structure based search for endonucleases that bind dsDNA in the A-like form and verification of their RNA/DNA cleaving activity. ( A ) DALI server ( 29 ) clustering of PD-(D/E)XK restriction endonucleases based on their structure. Recognition sequences are indicated, with bases bound in A-form according to the 3DNA program ( 31 ) in red. The percent cleavage of RNA and DNA strands is literature based ( 7 ). # For EcoRV and HinP1I the assignment was inconsistent between the structures. ( B ) Cleavage of dsDNA with a single target site for the indicated restriction endonucleases. The top DNA strand was labeled. The digestion pattern is as expected based on the location of cleavage sites. ( C ) Cleavage of RNA/DNA, the labeled top strand is DNA. ( D ) Cleavage of RNA/DNA, the labeled top strand is RNA. The sequence of substrates was in all cases the same except for T/U differences ( Supplementary Figure S15 ).
    Figure Legend Snippet: Structure based search for endonucleases that bind dsDNA in the A-like form and verification of their RNA/DNA cleaving activity. ( A ) DALI server ( 29 ) clustering of PD-(D/E)XK restriction endonucleases based on their structure. Recognition sequences are indicated, with bases bound in A-form according to the 3DNA program ( 31 ) in red. The percent cleavage of RNA and DNA strands is literature based ( 7 ). # For EcoRV and HinP1I the assignment was inconsistent between the structures. ( B ) Cleavage of dsDNA with a single target site for the indicated restriction endonucleases. The top DNA strand was labeled. The digestion pattern is as expected based on the location of cleavage sites. ( C ) Cleavage of RNA/DNA, the labeled top strand is DNA. ( D ) Cleavage of RNA/DNA, the labeled top strand is RNA. The sequence of substrates was in all cases the same except for T/U differences ( Supplementary Figure S15 ).

    Techniques Used: Activity Assay, Labeling, Sequencing

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0127321

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

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

    39) Product Images from "Morphological, Genome and Gene Expression Changes in Newly Induced Autopolyploid Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino"

    Article Title: Morphological, Genome and Gene Expression Changes in Newly Induced Autopolyploid Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17101690

    Representative variation in MSAP profiles. “→” to red arrows represents variation in DNA methylation between diploid and tetraploid plants; “+” represents fragments obtained after digestion with EcoR I or Hpa II/ Msp I; “−” represents fragments not digested by EcoR I or Hpa II/ Msp I; Type I fragments are nonmethylated and were presented in both the H ( EcoR I or Hpa II digest) and M ( EcoR I or Msp I digest) lanes; Type II are fully methylated and only appeared in the M lanes; Type III are hemimethylated and appeared in the H lanes; Type IV were fragments absent from both H and M lanes in diploid but present in either H or M lane of tetraploid, and vice versa.
    Figure Legend Snippet: Representative variation in MSAP profiles. “→” to red arrows represents variation in DNA methylation between diploid and tetraploid plants; “+” represents fragments obtained after digestion with EcoR I or Hpa II/ Msp I; “−” represents fragments not digested by EcoR I or Hpa II/ Msp I; Type I fragments are nonmethylated and were presented in both the H ( EcoR I or Hpa II digest) and M ( EcoR I or Msp I digest) lanes; Type II are fully methylated and only appeared in the M lanes; Type III are hemimethylated and appeared in the H lanes; Type IV were fragments absent from both H and M lanes in diploid but present in either H or M lane of tetraploid, and vice versa.

    Techniques Used: DNA Methylation Assay, Methylation

    40) Product Images from "The abundance of Fob1 modulates the efficiency of rRFBs to stall replication forks"

    Article Title: The abundance of Fob1 modulates the efficiency of rRFBs to stall replication forks

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx655

    Genetic map, 2D gel analysis of linear fragments corresponding to pYAC_MEM_3rRFBs+ and densitometry of the spots accumulated on the simple-Y arc. ( A ) Genetic map of pYAC_MEM_3rRFBs+ (8908 bp) showing its most relevant features (for further details see the legend of Figure 3 ). Note the insertion of an EcoRI fragment containing the three putative Fob1 binding sites expected to act as RFBs (indicated in red), described by Kobayashi ( 20 ). ( B ) Map of the 3708 bp linear fragment generated by digestion of pYAC_MEM_3rRFBs+ with SwaI and BamHI showing the relative position of the three putative RFBs. ( C ) Map of the 5186 bp linear fragment generated by digestion of pYAC_MEM_3rRFBs+ with EcoRV and MluI showing the relative position of the three putative RFBs. ( D ) 2D gel immunogram of the RIs corresponding to the SwaI-BamHI 3708 bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 5186-bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). The densitometric profile corresponding to the spots observed on the simple-Y arc shown in (D) is presented in ( H ) indicating the height of the peaks and the distance separating them.
    Figure Legend Snippet: Genetic map, 2D gel analysis of linear fragments corresponding to pYAC_MEM_3rRFBs+ and densitometry of the spots accumulated on the simple-Y arc. ( A ) Genetic map of pYAC_MEM_3rRFBs+ (8908 bp) showing its most relevant features (for further details see the legend of Figure 3 ). Note the insertion of an EcoRI fragment containing the three putative Fob1 binding sites expected to act as RFBs (indicated in red), described by Kobayashi ( 20 ). ( B ) Map of the 3708 bp linear fragment generated by digestion of pYAC_MEM_3rRFBs+ with SwaI and BamHI showing the relative position of the three putative RFBs. ( C ) Map of the 5186 bp linear fragment generated by digestion of pYAC_MEM_3rRFBs+ with EcoRV and MluI showing the relative position of the three putative RFBs. ( D ) 2D gel immunogram of the RIs corresponding to the SwaI-BamHI 3708 bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 5186-bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). The densitometric profile corresponding to the spots observed on the simple-Y arc shown in (D) is presented in ( H ) indicating the height of the peaks and the distance separating them.

    Techniques Used: Two-Dimensional Gel Electrophoresis, Binding Assay, Activated Clotting Time Assay, Generated

    Genetic map, 2D gel analysis of linear fragments corresponding to pYAC_AC_3′rRFBs+ and densitometry of the spots accumulated on the simple-Y arc. ( A ) Genetic map of pYAC_AC_3′rRFBs+ (8175 bp) showing its most relevant features (for further details see the legend of Figure 3 ). Note that here the three putative Fob1 binding sites expected to act as RFBs (indicated in red and white), described by Kobayashi ( 20 ), are equally distanced. ( B ) Map of the 3710-bp linear fragment generated by digestion of pYAC_AC_3′rRFBs+ with FspI and BamHI showing the relative position of the three putative RFBs. ( C ) Map of the 4454-bp linear fragment generated by digestion of pYAC_AC_3′rRFBs+ with EcoRV and MluI showing the relative position of the three putative RFBs. ( D ) 2D gel immunogram of the RIs corresponding to the FspI-BamHI 3710-bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 4454-bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). The densitometric profile corresponding to the spots observed on the simple-Y arc shown in (D) is presented in ( H ) indicating the height of the peaks and the distance separating them. For comparison, the densitometric profile corresponding to pYAC_MEM_3rRFBs shown in Figure 4H is presented on top.
    Figure Legend Snippet: Genetic map, 2D gel analysis of linear fragments corresponding to pYAC_AC_3′rRFBs+ and densitometry of the spots accumulated on the simple-Y arc. ( A ) Genetic map of pYAC_AC_3′rRFBs+ (8175 bp) showing its most relevant features (for further details see the legend of Figure 3 ). Note that here the three putative Fob1 binding sites expected to act as RFBs (indicated in red and white), described by Kobayashi ( 20 ), are equally distanced. ( B ) Map of the 3710-bp linear fragment generated by digestion of pYAC_AC_3′rRFBs+ with FspI and BamHI showing the relative position of the three putative RFBs. ( C ) Map of the 4454-bp linear fragment generated by digestion of pYAC_AC_3′rRFBs+ with EcoRV and MluI showing the relative position of the three putative RFBs. ( D ) 2D gel immunogram of the RIs corresponding to the FspI-BamHI 3710-bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 4454-bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). The densitometric profile corresponding to the spots observed on the simple-Y arc shown in (D) is presented in ( H ) indicating the height of the peaks and the distance separating them. For comparison, the densitometric profile corresponding to pYAC_MEM_3rRFBs shown in Figure 4H is presented on top.

    Techniques Used: Two-Dimensional Gel Electrophoresis, Binding Assay, Activated Clotting Time Assay, Generated

    Genetic map and 2D gel analysis of linear fragments corresponding to pYAC_MEM. ( A ) Genetic map of pYAC_MEM (7966 bp) showing its most relevant features: clockwise starting with the replication origin ARS4 (indicated in green), URA3 gene active in Saccharomyces cerevisiae (indicated in light blue), L1 lambda DNA used for hybridization (indicated in yellow), HIS3 gene active in S. cerevisiae (indicated in light blue), L2 lambda DNA used for hybridization (indicated in yellow), the ColE1 replication origin active only in Escherichia coli (indicated in gray), the ampicillin resistance gene active only in E. coli (indicated in gray) and the budding yeast centromeric sequence CEN6 (indicated in orange). The sites for specific restriction endonucleases are indicated outside the map. In addition, a magenta triangle points the position located 180° apart from the replication origin ARS4. ( B ) Map of the 2764-bp linear fragment generated by digestion of pYAC_MEM with SwaI and BamHI. ( C ) Map of the 4245-bp linear fragment generated by digestion of pYAC_MEM with EcoRV and MluI. ( D ) 2D gel immunogram of the RIs corresponding to the SwaI-BamHI 2764 bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 4245-bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). De-localized termination signals are indicated in magenta.
    Figure Legend Snippet: Genetic map and 2D gel analysis of linear fragments corresponding to pYAC_MEM. ( A ) Genetic map of pYAC_MEM (7966 bp) showing its most relevant features: clockwise starting with the replication origin ARS4 (indicated in green), URA3 gene active in Saccharomyces cerevisiae (indicated in light blue), L1 lambda DNA used for hybridization (indicated in yellow), HIS3 gene active in S. cerevisiae (indicated in light blue), L2 lambda DNA used for hybridization (indicated in yellow), the ColE1 replication origin active only in Escherichia coli (indicated in gray), the ampicillin resistance gene active only in E. coli (indicated in gray) and the budding yeast centromeric sequence CEN6 (indicated in orange). The sites for specific restriction endonucleases are indicated outside the map. In addition, a magenta triangle points the position located 180° apart from the replication origin ARS4. ( B ) Map of the 2764-bp linear fragment generated by digestion of pYAC_MEM with SwaI and BamHI. ( C ) Map of the 4245-bp linear fragment generated by digestion of pYAC_MEM with EcoRV and MluI. ( D ) 2D gel immunogram of the RIs corresponding to the SwaI-BamHI 2764 bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 4245-bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). De-localized termination signals are indicated in magenta.

    Techniques Used: Two-Dimensional Gel Electrophoresis, Lambda DNA Preparation, Hybridization, Sequencing, Generated

    Genetic map, 2D gel analysis of linear fragments corresponding to pYAC_AC_10rRFBs+ isolated from cells that overexpress Fob1 and densitometry of the spots accumulated on the simple-Y arc. ( A ) Genetic map of pYAC_AC_10rRFBs+ (8916 bp) showing its most relevant features (for further details see the legend of Figure 3 ). Note that here the fragment inserted at the unique SalI site of pYAC_MEM contained the 10 Fob1 binding sites described by Kobayashi ( 20 ) that were confirmed to act as RFBs in vivo (See Figures 4 and 5 ). ( B ) Map of the 3705-bp linear fragment generated by digestion of pYAC_AC_10rRFBs+ with SwaI and BamHI showing the relative position of the ten putative RFBs. ( C ) Map of the 5194-bp linear fragment generated by digestion of pYAC_AC_10rRFBs+ with EcoRV and MluI showing the relative position of the 10 putative RFBs. ( D ) 2D gel immunogram of the RIs corresponding to the SwaI-BamHI 3705 bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 5194 bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). The densitometric profile corresponding to the six most distal spots observed on the simple-Y arc shown in ( D ) is presented in ( H ) indicating the height of the peaks. For comparison, the densitometric profile corresponding to the 3705-bp SwaI-BamHI of pYAC_AC_10rRFBs isolated from the top2-td strain shown in Figure 4H is presented on top.
    Figure Legend Snippet: Genetic map, 2D gel analysis of linear fragments corresponding to pYAC_AC_10rRFBs+ isolated from cells that overexpress Fob1 and densitometry of the spots accumulated on the simple-Y arc. ( A ) Genetic map of pYAC_AC_10rRFBs+ (8916 bp) showing its most relevant features (for further details see the legend of Figure 3 ). Note that here the fragment inserted at the unique SalI site of pYAC_MEM contained the 10 Fob1 binding sites described by Kobayashi ( 20 ) that were confirmed to act as RFBs in vivo (See Figures 4 and 5 ). ( B ) Map of the 3705-bp linear fragment generated by digestion of pYAC_AC_10rRFBs+ with SwaI and BamHI showing the relative position of the ten putative RFBs. ( C ) Map of the 5194-bp linear fragment generated by digestion of pYAC_AC_10rRFBs+ with EcoRV and MluI showing the relative position of the 10 putative RFBs. ( D ) 2D gel immunogram of the RIs corresponding to the SwaI-BamHI 3705 bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 5194 bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). The densitometric profile corresponding to the six most distal spots observed on the simple-Y arc shown in ( D ) is presented in ( H ) indicating the height of the peaks. For comparison, the densitometric profile corresponding to the 3705-bp SwaI-BamHI of pYAC_AC_10rRFBs isolated from the top2-td strain shown in Figure 4H is presented on top.

    Techniques Used: Two-Dimensional Gel Electrophoresis, Isolation, Binding Assay, Activated Clotting Time Assay, In Vivo, Generated

    Genetic map, 2D gel analysis of linear fragments corresponding to pYAC_AC_10rRFBs+ and densitometry of the spots accumulated on the simple-Y arc. ( A ) Genetic map of pYAC_AC_10rRFBs+ (8916 bp) showing its most relevant features (for further details see the legend of Figure 3 ). Note that here the fragment inserted at the unique SalI site of pYAC_MEM contained the 10 Fob1 binding sites described by Kobayashi ( 20 ) that were confirmed to act as RFBs in vivo (See Figures 4 and 5 ). ( B ) Map of the 3705-bp linear fragment generated by digestion of pYAC_AC_10rRFBs+ with SwaI and BamHI showing the relative position of the 10 putative RFBs. ( C ) Map of the 5194-bp linear fragment generated by digestion of pYAC_AC_10rRFBs+ with EcoRV and MluI showing the relative position of the 10 putative RFBs. ( D ) 2D gel immunogram of the RIs corresponding to the SwaI-BamHI 3705-bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 5194-bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). The densitometric profile corresponding to the six most distal spots observed on the simple-Y arc shown in (D) is presented in ( H ) indicating the height of the peaks.
    Figure Legend Snippet: Genetic map, 2D gel analysis of linear fragments corresponding to pYAC_AC_10rRFBs+ and densitometry of the spots accumulated on the simple-Y arc. ( A ) Genetic map of pYAC_AC_10rRFBs+ (8916 bp) showing its most relevant features (for further details see the legend of Figure 3 ). Note that here the fragment inserted at the unique SalI site of pYAC_MEM contained the 10 Fob1 binding sites described by Kobayashi ( 20 ) that were confirmed to act as RFBs in vivo (See Figures 4 and 5 ). ( B ) Map of the 3705-bp linear fragment generated by digestion of pYAC_AC_10rRFBs+ with SwaI and BamHI showing the relative position of the 10 putative RFBs. ( C ) Map of the 5194-bp linear fragment generated by digestion of pYAC_AC_10rRFBs+ with EcoRV and MluI showing the relative position of the 10 putative RFBs. ( D ) 2D gel immunogram of the RIs corresponding to the SwaI-BamHI 3705-bp linear fragment with its diagrammatic interpretation in ( E ). The 2D gel immunogram of the RIs corresponding to the EcoRV-MluI 5194-bp linear fragment is shown in ( F ) with its diagrammatic interpretation in ( G ). The densitometric profile corresponding to the six most distal spots observed on the simple-Y arc shown in (D) is presented in ( H ) indicating the height of the peaks.

    Techniques Used: Two-Dimensional Gel Electrophoresis, Binding Assay, Activated Clotting Time Assay, In Vivo, Generated

    Related Articles

    Mutagenesis:

    Article Title: Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase
    Article Snippet: .. Therefore, to further confirm the mutation of hepP in PA14ΔhepP , we performed restriction enzyme digestion on the PCR products with EcoR V (New England Biolabs). ..

    Isolation:

    Article Title: Regulated Expression of the Beta2-Toxin Gene (cpb2) in Clostridium perfringens Type A Isolates from Horses with Gastrointestinal Diseases
    Article Snippet: .. Isolated C. perfringens DNA samples, prepared as previously described ( , ), were digested with EcoRV or HpaI (New England Biolabs), separated by electrophoresis on 1% agarose gels, and Southern transferred. .. The blots were hybridized with the DIG-labeled cpb2 probe, which was then detected using a DIG chemiluminescence detection system utilizing CSPD [disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2′-(5-chloro)tricyclo[3,3.1.13.7]decane}-4-yl)phenyl phosphate] ready-to-use substrate (Roche) as described earlier ( ).

    Purification:

    Article Title: A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting
    Article Snippet: .. The pGRFP plasmid was cut with EcoRV (NEB), 5′ dephosphorylated with CIAP, and gel purified. .. The linearized vector was blunt-end ligated to the SU66E20 BAC library inserts using T4 DNA ligase.

    Electrophoresis:

    Article Title: Integrative and Sequence Characteristics of a Novel Genetic Element, ICE6013, in Staphylococcus aureus
    Article Snippet: .. Genomic DNA was digested with EcoRV (New England Biolabs), and restriction fragments were separated by electrophoresis in 0.5% agarose. .. The fragments were blotted onto nylon membranes (Bio-Rad) by capillary transfer.

    Article Title: Regulated Expression of the Beta2-Toxin Gene (cpb2) in Clostridium perfringens Type A Isolates from Horses with Gastrointestinal Diseases
    Article Snippet: .. Isolated C. perfringens DNA samples, prepared as previously described ( , ), were digested with EcoRV or HpaI (New England Biolabs), separated by electrophoresis on 1% agarose gels, and Southern transferred. .. The blots were hybridized with the DIG-labeled cpb2 probe, which was then detected using a DIG chemiluminescence detection system utilizing CSPD [disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2′-(5-chloro)tricyclo[3,3.1.13.7]decane}-4-yl)phenyl phosphate] ready-to-use substrate (Roche) as described earlier ( ).

    Transgenic Assay:

    Article Title: Overexpression of Arabidopsis thaliana gibberellic acid 20 oxidase (AtGA20ox) gene enhance the vegetative growth and fiber quality in kenaf (Hibiscus cannabinus L.) plants
    Article Snippet: .. DNA from putative transgenic and UT plants was digested with EcoR V (NEB, UK). ..

    Molecular Weight:

    Article Title: Structural diversity of supercoiled DNA
    Article Snippet: .. BbvCI, EcoRV, Nb.BbvCI, NdeI, Nuclease Bal-31, T4 DNA Ligase, low molecular weight DNA ladder and 100 bp DNA ladder were purchased from New England Biolabs (Ipswich, MA). .. Proteinase K was purchased from Roche Molecular Biochemicals (Mannheim, Germany).

    Polymerase Chain Reaction:

    Article Title: Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase
    Article Snippet: .. Therefore, to further confirm the mutation of hepP in PA14ΔhepP , we performed restriction enzyme digestion on the PCR products with EcoR V (New England Biolabs). ..

    Plasmid Preparation:

    Article Title: A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting
    Article Snippet: .. The pGRFP plasmid was cut with EcoRV (NEB), 5′ dephosphorylated with CIAP, and gel purified. .. The linearized vector was blunt-end ligated to the SU66E20 BAC library inserts using T4 DNA ligase.

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    Southern blotting of <t>EcoRV-digested</t> genomic <t>DNA</t> using an ICE 6013 probe. Lanes 1 and 11, DIG-labeled HindIII-digested lambda ladder (Roche); lanes 2 and 10, blank; lanes 3 to 9, seven strains. The arrow indicates the expected 9.6-kb fragment from strain HDG2.
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    Southern blotting of EcoRV-digested genomic DNA using an ICE 6013 probe. Lanes 1 and 11, DIG-labeled HindIII-digested lambda ladder (Roche); lanes 2 and 10, blank; lanes 3 to 9, seven strains. The arrow indicates the expected 9.6-kb fragment from strain HDG2.

    Journal: Journal of Bacteriology

    Article Title: Integrative and Sequence Characteristics of a Novel Genetic Element, ICE6013, in Staphylococcus aureus

    doi: 10.1128/JB.00352-09

    Figure Lengend Snippet: Southern blotting of EcoRV-digested genomic DNA using an ICE 6013 probe. Lanes 1 and 11, DIG-labeled HindIII-digested lambda ladder (Roche); lanes 2 and 10, blank; lanes 3 to 9, seven strains. The arrow indicates the expected 9.6-kb fragment from strain HDG2.

    Article Snippet: Genomic DNA was digested with EcoRV (New England Biolabs), and restriction fragments were separated by electrophoresis in 0.5% agarose.

    Techniques: Southern Blot, Labeling

    RFLP-Southern blot analysis of HpaI- or EcoRV-digested DNA from horse GI disease isolates. Total DNA isolated from each of the specified C. perfringens strains was digested with HpaI (A) or EcoRV (B) and then Southern transferred. The Southern blots were

    Journal: Journal of Clinical Microbiology

    Article Title: Regulated Expression of the Beta2-Toxin Gene (cpb2) in Clostridium perfringens Type A Isolates from Horses with Gastrointestinal Diseases

    doi: 10.1128/JCM.43.8.4002-4009.2005

    Figure Lengend Snippet: RFLP-Southern blot analysis of HpaI- or EcoRV-digested DNA from horse GI disease isolates. Total DNA isolated from each of the specified C. perfringens strains was digested with HpaI (A) or EcoRV (B) and then Southern transferred. The Southern blots were

    Article Snippet: Isolated C. perfringens DNA samples, prepared as previously described ( , ), were digested with EcoRV or HpaI (New England Biolabs), separated by electrophoresis on 1% agarose gels, and Southern transferred.

    Techniques: Southern Blot, Isolation

    Controlled release of USPIO micelles by environmental triggers . (A) Self-assembly of EcoRV-sensitive ssDNA-USPIO clusters, and subsequent enzymatic treatment results in measurable changes in R 2 relaxation coefficient relative to initial values. Following EcoRV treatment, R 2 values return to baseline, a phenomenon that is in significant contrast to the effects of EcoRI treatment of the same clusters (n = 6). * p

    Journal: Journal of Nanobiotechnology

    Article Title: Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs)

    doi: 10.1186/1477-3155-9-7

    Figure Lengend Snippet: Controlled release of USPIO micelles by environmental triggers . (A) Self-assembly of EcoRV-sensitive ssDNA-USPIO clusters, and subsequent enzymatic treatment results in measurable changes in R 2 relaxation coefficient relative to initial values. Following EcoRV treatment, R 2 values return to baseline, a phenomenon that is in significant contrast to the effects of EcoRI treatment of the same clusters (n = 6). * p

    Article Snippet: The restriction enzymes EcoRI and EcoRV were purchased from New England Biolabs (Ipswich, MA).

    Techniques:

    Confirmation of the mutation in PA14Δ hepP . PA14 and PA14Δ hepP were grown in LB broth and the chromosomal DNA was extracted. a PCR analysis to detect the presence of MAR2xT7 within hepP . PCR reactions were run using the chromosomal DNA from each strain as a template and primers corresponding to the DNA sequences 94 bp upstream and 179 bp downstream of the hepP structural gene ( zbdP- For3/ hepP- Rev3, Table 2 ). The expected 1926-bp fragment from PA14 (lane 1) and the 2920-bp fragment (the additional 994 bp from MAR2xT7 ) from PA14Δ hepP (lane 2) were detected. Lane 3 is a no-template control and the molecular size standards are in lane 4. b Restriction analysis of the PCR products. The coding sequence for hepP does not contain an EcoR V restriction enzyme site, while MAR2xT7 contains a single EcoR V site. Digestion of the PCR products with EcoR V failed to reduce the size of the 1926-bp fragment obtained from PA14 (lane 3) but resulted in the cleavage of the product obtained from PA14Δ hepP into the expected 800 bp and 2120 bp fragments (lane 4). Lane 1 contains the molecular size standards; lane 2 was left empty

    Journal: BMC Microbiology

    Article Title: Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase

    doi: 10.1186/s12866-017-1141-0

    Figure Lengend Snippet: Confirmation of the mutation in PA14Δ hepP . PA14 and PA14Δ hepP were grown in LB broth and the chromosomal DNA was extracted. a PCR analysis to detect the presence of MAR2xT7 within hepP . PCR reactions were run using the chromosomal DNA from each strain as a template and primers corresponding to the DNA sequences 94 bp upstream and 179 bp downstream of the hepP structural gene ( zbdP- For3/ hepP- Rev3, Table 2 ). The expected 1926-bp fragment from PA14 (lane 1) and the 2920-bp fragment (the additional 994 bp from MAR2xT7 ) from PA14Δ hepP (lane 2) were detected. Lane 3 is a no-template control and the molecular size standards are in lane 4. b Restriction analysis of the PCR products. The coding sequence for hepP does not contain an EcoR V restriction enzyme site, while MAR2xT7 contains a single EcoR V site. Digestion of the PCR products with EcoR V failed to reduce the size of the 1926-bp fragment obtained from PA14 (lane 3) but resulted in the cleavage of the product obtained from PA14Δ hepP into the expected 800 bp and 2120 bp fragments (lane 4). Lane 1 contains the molecular size standards; lane 2 was left empty

    Article Snippet: Therefore, to further confirm the mutation of hepP in PA14ΔhepP , we performed restriction enzyme digestion on the PCR products with EcoR V (New England Biolabs).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Sequencing