bglii  (New England Biolabs)


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    Name:
    BglII
    Description:
    BglII 10 000 units
    Catalog Number:
    r0144l
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    249
    Size:
    10 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs bglii
    BglII
    BglII 10 000 units
    https://www.bioz.com/result/bglii/product/New England Biolabs
    Average 99 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    bglii - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Development of an Intergenotypic Hepatitis C Virus (HCV) Cell Culture Method To Assess Antiviral Susceptibilities and Resistance Development of HCV NS3 Protease Genes from HCV Genotypes 1 to 6 ▿"

    Article Title: Development of an Intergenotypic Hepatitis C Virus (HCV) Cell Culture Method To Assess Antiviral Susceptibilities and Resistance Development of HCV NS3 Protease Genes from HCV Genotypes 1 to 6 ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02698-09

    Replication kinetics in Huh7.5 cells of the parental JFH1 and Jc1 full-length RNA transcripts and comparison with that of Jc1-BB containing introduced BstBI and BglII restriction sites in NS3 for protease gene insertion. The replication-defective mutant JFH1-GND was used as a negative control. The y axis records the percentage of HCV NS5A-positive cells scored by fluorescence microscopy.
    Figure Legend Snippet: Replication kinetics in Huh7.5 cells of the parental JFH1 and Jc1 full-length RNA transcripts and comparison with that of Jc1-BB containing introduced BstBI and BglII restriction sites in NS3 for protease gene insertion. The replication-defective mutant JFH1-GND was used as a negative control. The y axis records the percentage of HCV NS5A-positive cells scored by fluorescence microscopy.

    Techniques Used: Mutagenesis, Negative Control, Fluorescence, Microscopy

    2) Product Images from "Sp1 Regulates Chromatin Looping between an Intronic Enhancer and Distal Promoter of the Human Heme Oxygenase-1 Gene in Renal Cells *"

    Article Title: Sp1 Regulates Chromatin Looping between an Intronic Enhancer and Distal Promoter of the Human Heme Oxygenase-1 Gene in Renal Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.058586

    Confirmation of chromatin looping by 3C assay with BglII and/or ApaLI restriction enzyme. A , schematic showing potential interaction between the fragments containing HS-2 region and the internal enhancer ( square ) and transcription factor(s) ( TF ). B , BglII
    Figure Legend Snippet: Confirmation of chromatin looping by 3C assay with BglII and/or ApaLI restriction enzyme. A , schematic showing potential interaction between the fragments containing HS-2 region and the internal enhancer ( square ) and transcription factor(s) ( TF ). B , BglII

    Techniques Used:

    3) Product Images from "Simple and Cost-Effective Restriction Endonuclease Analysis of Human Adenoviruses"

    Article Title: Simple and Cost-Effective Restriction Endonuclease Analysis of Human Adenoviruses

    Journal: BioMed Research International

    doi: 10.1155/2014/363790

    Picture of the REA. (a) REA pattern for BamHI and SmaI (fast digest). (b) REA pattern for BglII and HindIII . M shows Lambda DNA-HindIII digest marker. Numbers 1, 3, 4, and 37 show HAdV-1, -3, -4, and -37, respectively.
    Figure Legend Snippet: Picture of the REA. (a) REA pattern for BamHI and SmaI (fast digest). (b) REA pattern for BglII and HindIII . M shows Lambda DNA-HindIII digest marker. Numbers 1, 3, 4, and 37 show HAdV-1, -3, -4, and -37, respectively.

    Techniques Used: Lambda DNA Preparation, Marker

    4) 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

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202138

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

    Techniques Used: Electrophoresis, Sequencing

    6) Product Images from "Examination and Reconstruction of Three Ancient Endogenous Parvovirus Capsid Protein Gene Remnants Found in Rodent Genomes"

    Article Title: Examination and Reconstruction of Three Ancient Endogenous Parvovirus Capsid Protein Gene Remnants Found in Rodent Genomes

    Journal: Journal of Virology

    doi: 10.1128/JVI.01542-18

    Expression and assembly of chimeras constructed by combining BglII and PhoI restriction enzyme digestions of the R. norvegicus EVE with those of CPV-2. (A) Western blot showing capsid proteins chimeras or CPV-2 expressed in insect cells and stained with a polyclonal anti-CPV antibody. (B) Electron micrograph of CPV-2 VLPs showing assembled capsids. (C to H) Electron micrographs of chimeras between the R. norvegicus EVE and CPV-2, showing that capsid proteins do not assemble. Diagrams of the chimeras are also shown above each micrograph, with the VP2 gene divided at the BglII and PhoI restriction enzyme sites and the R. norvegicus EVE represented in green.
    Figure Legend Snippet: Expression and assembly of chimeras constructed by combining BglII and PhoI restriction enzyme digestions of the R. norvegicus EVE with those of CPV-2. (A) Western blot showing capsid proteins chimeras or CPV-2 expressed in insect cells and stained with a polyclonal anti-CPV antibody. (B) Electron micrograph of CPV-2 VLPs showing assembled capsids. (C to H) Electron micrographs of chimeras between the R. norvegicus EVE and CPV-2, showing that capsid proteins do not assemble. Diagrams of the chimeras are also shown above each micrograph, with the VP2 gene divided at the BglII and PhoI restriction enzyme sites and the R. norvegicus EVE represented in green.

    Techniques Used: Expressing, Construct, Western Blot, Staining

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

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

    Journal: mBio

    doi: 10.1128/mBio.00173-15

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

    Techniques Used: Isolation, Methylation

    8) Product Images from "Are Immune Modulating Single Nucleotide Polymorphisms Associated with Necrotizing Enterocolitis?"

    Article Title: Are Immune Modulating Single Nucleotide Polymorphisms Associated with Necrotizing Enterocolitis?

    Journal: Scientific Reports

    doi: 10.1038/srep18369

    PCR Amplification and BglII endonuclease digestion ( a ) PCR amplification of TRIM21 rs660 with a product at 420 bp. ( b ) Rs660 there is a nucleotide alteration from T to C that will alter the site of BglII endonuclease digestion with resultant products at 255 and 165 bp.
    Figure Legend Snippet: PCR Amplification and BglII endonuclease digestion ( a ) PCR amplification of TRIM21 rs660 with a product at 420 bp. ( b ) Rs660 there is a nucleotide alteration from T to C that will alter the site of BglII endonuclease digestion with resultant products at 255 and 165 bp.

    Techniques Used: Polymerase Chain Reaction, Amplification

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202138

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

    Techniques Used: Electrophoresis, Sequencing

    10) Product Images from "Development of an Intergenotypic Hepatitis C Virus (HCV) Cell Culture Method To Assess Antiviral Susceptibilities and Resistance Development of HCV NS3 Protease Genes from HCV Genotypes 1 to 6 ▿"

    Article Title: Development of an Intergenotypic Hepatitis C Virus (HCV) Cell Culture Method To Assess Antiviral Susceptibilities and Resistance Development of HCV NS3 Protease Genes from HCV Genotypes 1 to 6 ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02698-09

    Replication kinetics in Huh7.5 cells of the parental JFH1 and Jc1 full-length RNA transcripts and comparison with that of Jc1-BB containing introduced BstBI and BglII restriction sites in NS3 for protease gene insertion. The replication-defective mutant JFH1-GND was used as a negative control. The y axis records the percentage of HCV NS5A-positive cells scored by fluorescence microscopy.
    Figure Legend Snippet: Replication kinetics in Huh7.5 cells of the parental JFH1 and Jc1 full-length RNA transcripts and comparison with that of Jc1-BB containing introduced BstBI and BglII restriction sites in NS3 for protease gene insertion. The replication-defective mutant JFH1-GND was used as a negative control. The y axis records the percentage of HCV NS5A-positive cells scored by fluorescence microscopy.

    Techniques Used: Mutagenesis, Negative Control, Fluorescence, Microscopy

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

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

    Journal: mBio

    doi: 10.1128/mBio.00173-15

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

    Techniques Used: Isolation, Methylation

    12) Product Images from "Histone H3 Localizes to the Centromeric DNA in Budding Yeast"

    Article Title: Histone H3 Localizes to the Centromeric DNA in Budding Yeast

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002739

    Models of how H3 and Cse4 can co-occupy the centromeric DNA. A heterotetramer of H3, H2A, H2B and H4 is colored in green and a heterotetramer containing Cse4 instead of H3 is blue.1) A heterotypic octamer containing both Cse4 and H3. 2) A heterotypic octamer with additional Cse4 bound to it. 3) A Cse4 hemisome incorporated in the loop of a conventional nucleosome. A DNA fragment of 207 bp is sufficient to accommodate this arrangement (without spacer DNA). 4) Two conventional nucleosomes flanking a Cse4 hemisome. The scissors indicate the BglII sites flanking the 214 bp fragment excised in our experiment. In case of model 4 this fragment would be tethered to non-centromeric DNA. The tethering was not observed in our experiments ( Figure 1C ). See text for discussion and additional details.
    Figure Legend Snippet: Models of how H3 and Cse4 can co-occupy the centromeric DNA. A heterotetramer of H3, H2A, H2B and H4 is colored in green and a heterotetramer containing Cse4 instead of H3 is blue.1) A heterotypic octamer containing both Cse4 and H3. 2) A heterotypic octamer with additional Cse4 bound to it. 3) A Cse4 hemisome incorporated in the loop of a conventional nucleosome. A DNA fragment of 207 bp is sufficient to accommodate this arrangement (without spacer DNA). 4) Two conventional nucleosomes flanking a Cse4 hemisome. The scissors indicate the BglII sites flanking the 214 bp fragment excised in our experiment. In case of model 4 this fragment would be tethered to non-centromeric DNA. The tethering was not observed in our experiments ( Figure 1C ). See text for discussion and additional details.

    Techniques Used:

    Composition of the centromeric nucleosome. A) The CEN-containing minichromosomes can be specifically co-immunoprecipitated with Cse4 and H3. Lysates from strains transformed with the minichromosomes 1021 (wt), 1498 (Cse4-HA6) and 1407 (H3-HA3) were incubated with anti-HA antibody and Dynabeads. DNA was eluted off the beads and separated on a 1% agarose gel. Southern blot was analyzed using a 32 P labeled TRP1 probe. The map of the minichromosome is shown in Figure S1 . B) Experimental setup for the immunoprecipitation of minichromosomes digested with restriction enzyme. Chromatin is digested with BglII and incubated with anti-HA antibody recognizing tagged histones and protein A Dynabeads. Minichromosome digest with BglII produces three different fragments: a linearized full-length minichromosome (1), a CEN-less fragment (2) which can be detected with TRP1 probe and a small CEN fragment (3) which can be detected with an LNA oligonucleotide. The red ellipse is depicting the centromeric nucleosome. C) Cse4 binding is restricted to minichromosomal CEN DNA. BglII-treated chromatin of strains carrying the minichromosome with BglII restriction sites 50 bp upstream and downstream of CEN boundaries was immunoprecipitated with anti-HA antibody. The strains were 1498 (Cse4-HA6), 1577 (H4-HA3), 1576 (H2A-HA3), 1587 (H2B-HA3), 1407 (H3-HA3), 1593 (Scm3-HA6), and 1021 (wt). DNA was analyzed as in (A) with 32 P labeled TRP1 probe. D) H3 is associated with the CEN DNA. Top: Scheme of the excised CEN fragment. Double-DIG labeled LNA probe for CDEI/II is indicated. Bottom: Immunoprecipitated DNA from experiments shown in (C) was separated on a 6% denaturing TBE polyacrylamide gel. Southern blot was analyzed using a double-DIG labeled LNA probe for CDEI/II. Western blots showing immunoprecipitation of the tagged proteins are shown in Figure S4A .
    Figure Legend Snippet: Composition of the centromeric nucleosome. A) The CEN-containing minichromosomes can be specifically co-immunoprecipitated with Cse4 and H3. Lysates from strains transformed with the minichromosomes 1021 (wt), 1498 (Cse4-HA6) and 1407 (H3-HA3) were incubated with anti-HA antibody and Dynabeads. DNA was eluted off the beads and separated on a 1% agarose gel. Southern blot was analyzed using a 32 P labeled TRP1 probe. The map of the minichromosome is shown in Figure S1 . B) Experimental setup for the immunoprecipitation of minichromosomes digested with restriction enzyme. Chromatin is digested with BglII and incubated with anti-HA antibody recognizing tagged histones and protein A Dynabeads. Minichromosome digest with BglII produces three different fragments: a linearized full-length minichromosome (1), a CEN-less fragment (2) which can be detected with TRP1 probe and a small CEN fragment (3) which can be detected with an LNA oligonucleotide. The red ellipse is depicting the centromeric nucleosome. C) Cse4 binding is restricted to minichromosomal CEN DNA. BglII-treated chromatin of strains carrying the minichromosome with BglII restriction sites 50 bp upstream and downstream of CEN boundaries was immunoprecipitated with anti-HA antibody. The strains were 1498 (Cse4-HA6), 1577 (H4-HA3), 1576 (H2A-HA3), 1587 (H2B-HA3), 1407 (H3-HA3), 1593 (Scm3-HA6), and 1021 (wt). DNA was analyzed as in (A) with 32 P labeled TRP1 probe. D) H3 is associated with the CEN DNA. Top: Scheme of the excised CEN fragment. Double-DIG labeled LNA probe for CDEI/II is indicated. Bottom: Immunoprecipitated DNA from experiments shown in (C) was separated on a 6% denaturing TBE polyacrylamide gel. Southern blot was analyzed using a double-DIG labeled LNA probe for CDEI/II. Western blots showing immunoprecipitation of the tagged proteins are shown in Figure S4A .

    Techniques Used: Immunoprecipitation, Transformation Assay, Incubation, Agarose Gel Electrophoresis, Southern Blot, Labeling, Binding Assay, Western Blot

    Histone H3 localizes to the centromeric DNA. A) H3 is associated with CEN DNA throughout the cell cycle. Strains carrying the minichromosomes with BglII restriction sites 50 bp upstream and downstream of CEN boundaries, 1498 (Cse4-HA6), 1407 (H3-HA3), and 1587 (H2B-HA3) were arrested in G1 with alpha factor and in G2 with nocodazole/benomyl. Chromatin was treated with BglII and immunoprecipitated with anti-HA antibody. DNA was eluted off the beads and resolved on a 6% denaturing TBE polyacrylamide gel. Southern blot was analyzed with a double-DIG labeled LNA probe for CDEI/II. The FACS profiles are shown in Figure S4B . B) H3 is associated with the CEN DNA on a native chromosome IV. BglII-treated chromatin of strains with BglII sites 50 bp upstream and downstream of CEN boundaries on chromosome IV 2059 (wt), 2043 (Cse4-HA3), and 2042 (H3-HA3) was immunoprecipitated with anti-HA antibody. DNA was eluted off the beads, separated on a 6% denaturing TBE polyacrylamide gel and analyzed with a double-DIG labeled LNA probe for CDEI/II. C) Minichromosome-bound histone H3 does not turn over during the immunoprecipitation procedure. Lysates of strains 1021 (wt, carrying the minichromosome), 1407 (H3-HA, carrying the minichromosome), 1407 (H3-HA3, without the minichromosome), and mixed lysate of 1021 (wt with minichromosome) and 1407 (H3-HA3, without the minichromosome) were incubated with anti-HA antibody and Dynabeads. DNA was eluted off the beads, separated on a 1% agarose gel and analyzed using a 32 P labeled TRP1 probe.
    Figure Legend Snippet: Histone H3 localizes to the centromeric DNA. A) H3 is associated with CEN DNA throughout the cell cycle. Strains carrying the minichromosomes with BglII restriction sites 50 bp upstream and downstream of CEN boundaries, 1498 (Cse4-HA6), 1407 (H3-HA3), and 1587 (H2B-HA3) were arrested in G1 with alpha factor and in G2 with nocodazole/benomyl. Chromatin was treated with BglII and immunoprecipitated with anti-HA antibody. DNA was eluted off the beads and resolved on a 6% denaturing TBE polyacrylamide gel. Southern blot was analyzed with a double-DIG labeled LNA probe for CDEI/II. The FACS profiles are shown in Figure S4B . B) H3 is associated with the CEN DNA on a native chromosome IV. BglII-treated chromatin of strains with BglII sites 50 bp upstream and downstream of CEN boundaries on chromosome IV 2059 (wt), 2043 (Cse4-HA3), and 2042 (H3-HA3) was immunoprecipitated with anti-HA antibody. DNA was eluted off the beads, separated on a 6% denaturing TBE polyacrylamide gel and analyzed with a double-DIG labeled LNA probe for CDEI/II. C) Minichromosome-bound histone H3 does not turn over during the immunoprecipitation procedure. Lysates of strains 1021 (wt, carrying the minichromosome), 1407 (H3-HA, carrying the minichromosome), 1407 (H3-HA3, without the minichromosome), and mixed lysate of 1021 (wt with minichromosome) and 1407 (H3-HA3, without the minichromosome) were incubated with anti-HA antibody and Dynabeads. DNA was eluted off the beads, separated on a 1% agarose gel and analyzed using a 32 P labeled TRP1 probe.

    Techniques Used: Immunoprecipitation, Southern Blot, Labeling, FACS, Incubation, Agarose Gel Electrophoresis

    Cse4 association with CDEI/II and CDEIII. A) Cse4 nucleosome straddles the boundary between CDEII and CDEIII. Left: Map of the minichromosome utilized in the experiment. The construct contains 850 bp of pericentromeric sequence of chromosome IV, TRP1 marker, ARS1 and pUC19 sequence and has a size of 4.5 kb. There are two BglII sites: between CDEII and CDEIII in the CEN and in the ARS1 . Right: BglII-treated chromatin of a strain 1498 (Cse4-HA6) carrying the minichromosome was immunoprecipitated with anti-HA antibody. DNA was eluted off the beads and separated on a 1% agarose gel. Southern blot was analyzed with a 32 P labeled probe for the pericentric CEN4 sequence (to detect the CDEI/II containing fragment) and a 32 P labeled probe for the TRP1 gene (to detect the CDEIII containing fragment). B) Both Cse4 and H3 are associated with the CDEI/II fragment. Left: Scheme of CDEI/II fragment excised from the minichromosome. Double-DIG labeled LNA probe for CDEI/II is indicated. Right: BglII-treated chromatin of strain 1498 (Cse4-HA6) and 1407 (H3-HA3) carrying the minichromosome with BglII sites between CDEII and CDEIII and 50 bp upstream of CDEI was cross-linked with formaldehyde and immunoprecipitated with anti-HA antibody. DNA was eluted off the beads and resolved on a 6% denaturing TBE polyacrylamide gel. Southern blot was analyzed with a double-DIG labeled LNA probe for CDEI/II. C) Both the CDEI/II and the CDEIII fragments can be co-immunoprecipitated with Cse4 and H3. Strains 1021 (wt), 1407 (H3-HA3), and 1498 (Cse4-HA6) carried the minichromosome where either the CDEI/II (left) or the CDEIII fragment (right) was flanked with BglII sites. BglII-treated chromatin was either not cross-linked or cross-linked with formaldehyde and immunoprecipitated with anti-HA antibody. The immunoprecipitated DNA was purified, size fractionated, and subjected to qPCR analysis. Bar graphs represent the average values from several independent experiments with SDs.
    Figure Legend Snippet: Cse4 association with CDEI/II and CDEIII. A) Cse4 nucleosome straddles the boundary between CDEII and CDEIII. Left: Map of the minichromosome utilized in the experiment. The construct contains 850 bp of pericentromeric sequence of chromosome IV, TRP1 marker, ARS1 and pUC19 sequence and has a size of 4.5 kb. There are two BglII sites: between CDEII and CDEIII in the CEN and in the ARS1 . Right: BglII-treated chromatin of a strain 1498 (Cse4-HA6) carrying the minichromosome was immunoprecipitated with anti-HA antibody. DNA was eluted off the beads and separated on a 1% agarose gel. Southern blot was analyzed with a 32 P labeled probe for the pericentric CEN4 sequence (to detect the CDEI/II containing fragment) and a 32 P labeled probe for the TRP1 gene (to detect the CDEIII containing fragment). B) Both Cse4 and H3 are associated with the CDEI/II fragment. Left: Scheme of CDEI/II fragment excised from the minichromosome. Double-DIG labeled LNA probe for CDEI/II is indicated. Right: BglII-treated chromatin of strain 1498 (Cse4-HA6) and 1407 (H3-HA3) carrying the minichromosome with BglII sites between CDEII and CDEIII and 50 bp upstream of CDEI was cross-linked with formaldehyde and immunoprecipitated with anti-HA antibody. DNA was eluted off the beads and resolved on a 6% denaturing TBE polyacrylamide gel. Southern blot was analyzed with a double-DIG labeled LNA probe for CDEI/II. C) Both the CDEI/II and the CDEIII fragments can be co-immunoprecipitated with Cse4 and H3. Strains 1021 (wt), 1407 (H3-HA3), and 1498 (Cse4-HA6) carried the minichromosome where either the CDEI/II (left) or the CDEIII fragment (right) was flanked with BglII sites. BglII-treated chromatin was either not cross-linked or cross-linked with formaldehyde and immunoprecipitated with anti-HA antibody. The immunoprecipitated DNA was purified, size fractionated, and subjected to qPCR analysis. Bar graphs represent the average values from several independent experiments with SDs.

    Techniques Used: Construct, Sequencing, Marker, Immunoprecipitation, Agarose Gel Electrophoresis, Southern Blot, Labeling, Purification, Real-time Polymerase Chain Reaction

    Co-occupancy of the centromeric DNA by histone H3 and Cse4. A) Only the 214 bp BglII CEN4 fragment and no full-length minichromosome is detected in the ChIP/qPCR assay. DNA isolated from untreated and BglII-treated lysates was size-fractionated on 2% agarose gel and analyzed by qPCR. A PCR product after 30 cycles of amplification in a conventional PCR reaction with the same primers that were used for qPCR is shown below. B) Minichromosomal CEN DNA can be co-immunoprecipitated with H3 and Cse4. BglII-treated chromatin of the strains 1021 (wt), 1407 (H3-HA3), and 1498 (Cse4-HA6) carrying the minichromosome was either not cross-linked or cross-linked with formaldehyde and immunoprecipitated with anti-HA antibody. The immunoprecipitated DNA was purified and size fractionated and subjected to qPCR analysis. C) CEN DNA of the native chromosome IV can be co-immunoprecipitatd with H3 and Cse4. BglII-treated chromatin of the strains 2059 (wt), 2042 (H3-HA3), and 2043 (Cse4-HA6) with CEN DNA of the native chromosome IV flanked with BglII was either not cross-linked or cross-linked with formaldehyde followed by immunoprecipitation as in (B). D) Flowchart of the sequential Cse4-H3 ChIP. E) Sequential ChIP of minichromosomal CEN DNA. BglII-treated chromatin of the strains 1923 (Cse4-Myc6) and 2300 (H3-HA3, Cse4-Myc6) carrying the minichromosome was cross-linked with formaldehyde and immunoprecipitated with anti-Myc or anti-HA antibody as indicated in the figure, the DNA was eluted off the beads and re-immunoprecipitated with anti-HA antibody. The immunoprecipitated DNA was purified, size fractionated on a 2% agarose gel and subjected to qPCR analysis. F) The same as in (E) but performed with the native CEN DNA. The strains, 2562 (Cse4-Myc6), and 2561 (H3-HA3, Cse4-Myc6) had CEN DNA of the native chromosome IV flanked with BglII. The bar graphs represent the average values from several independent experiments with SDs.
    Figure Legend Snippet: Co-occupancy of the centromeric DNA by histone H3 and Cse4. A) Only the 214 bp BglII CEN4 fragment and no full-length minichromosome is detected in the ChIP/qPCR assay. DNA isolated from untreated and BglII-treated lysates was size-fractionated on 2% agarose gel and analyzed by qPCR. A PCR product after 30 cycles of amplification in a conventional PCR reaction with the same primers that were used for qPCR is shown below. B) Minichromosomal CEN DNA can be co-immunoprecipitated with H3 and Cse4. BglII-treated chromatin of the strains 1021 (wt), 1407 (H3-HA3), and 1498 (Cse4-HA6) carrying the minichromosome was either not cross-linked or cross-linked with formaldehyde and immunoprecipitated with anti-HA antibody. The immunoprecipitated DNA was purified and size fractionated and subjected to qPCR analysis. C) CEN DNA of the native chromosome IV can be co-immunoprecipitatd with H3 and Cse4. BglII-treated chromatin of the strains 2059 (wt), 2042 (H3-HA3), and 2043 (Cse4-HA6) with CEN DNA of the native chromosome IV flanked with BglII was either not cross-linked or cross-linked with formaldehyde followed by immunoprecipitation as in (B). D) Flowchart of the sequential Cse4-H3 ChIP. E) Sequential ChIP of minichromosomal CEN DNA. BglII-treated chromatin of the strains 1923 (Cse4-Myc6) and 2300 (H3-HA3, Cse4-Myc6) carrying the minichromosome was cross-linked with formaldehyde and immunoprecipitated with anti-Myc or anti-HA antibody as indicated in the figure, the DNA was eluted off the beads and re-immunoprecipitated with anti-HA antibody. The immunoprecipitated DNA was purified, size fractionated on a 2% agarose gel and subjected to qPCR analysis. F) The same as in (E) but performed with the native CEN DNA. The strains, 2562 (Cse4-Myc6), and 2561 (H3-HA3, Cse4-Myc6) had CEN DNA of the native chromosome IV flanked with BglII. The bar graphs represent the average values from several independent experiments with SDs.

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Isolation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Immunoprecipitation, Purification

    13) Product Images from "Restriction Cascade Exponential Amplification (RCEA) assay with an attomolar detection limit: a novel, highly specific, isothermal alternative to qPCR"

    Article Title: Restriction Cascade Exponential Amplification (RCEA) assay with an attomolar detection limit: a novel, highly specific, isothermal alternative to qPCR

    Journal: Scientific Reports

    doi: 10.1038/srep07737

    General schematic of the assay used for REase conjugate testing. (a) An oligonucleotide probe specific for a target of interest is conjugated to a mutant REase (either the S17C BamHI or K249C EcoRI), and attached to a solid surface using biotin. An oligonucleotide target complementary to the probe is added, and it hybridizes to the probe. (b) The hybrid is specifically cleaved by a free REase BglII that is added to the reaction solution. The mutant REase is subsequently released into the solution. (c) The reaction solution is transferred to a chamber containing an HRP conjugate immobilized through a dsDNA linker containing the recognition sequence of the mutant REase. The addition of the released REase generated in (b) catalyzes the linker cleavage and HRP release. (d) The released HRP is quantified colorimetrically.
    Figure Legend Snippet: General schematic of the assay used for REase conjugate testing. (a) An oligonucleotide probe specific for a target of interest is conjugated to a mutant REase (either the S17C BamHI or K249C EcoRI), and attached to a solid surface using biotin. An oligonucleotide target complementary to the probe is added, and it hybridizes to the probe. (b) The hybrid is specifically cleaved by a free REase BglII that is added to the reaction solution. The mutant REase is subsequently released into the solution. (c) The reaction solution is transferred to a chamber containing an HRP conjugate immobilized through a dsDNA linker containing the recognition sequence of the mutant REase. The addition of the released REase generated in (b) catalyzes the linker cleavage and HRP release. (d) The released HRP is quantified colorimetrically.

    Techniques Used: Mutagenesis, Sequencing, Generated

    Effects of the target sequence alteration and addition of foreign DNA on performance of the RCEA assay employing the K249C EcoRI system. The X-axis shows the experimental conditions: AMC, the target type (BG-40 is fully cognate; 12/40 contains 12 cognate nucleotides centered around the restriction site and non-cognate ends, Table 1 ); gDNA, presence or absence of mouse genomic DNA (80 ng per reaction); Wash, whether the beads carrying immobilized Ramp conjugates were washed after target hybridization prior to addition of the Rrec BglII. The Y-axis shows the background-subtracted HRP signal values (with the background calculated as the mean signal generated for zero target concentrations). For normalization and comparison of sample series, the HRP signal values were expressed as the percentages of the maximum background-subtracted OD 655 obtained for the fully cognate target (the left-most bar of the graph). For all test samples, the target concentrations were 1 pM.
    Figure Legend Snippet: Effects of the target sequence alteration and addition of foreign DNA on performance of the RCEA assay employing the K249C EcoRI system. The X-axis shows the experimental conditions: AMC, the target type (BG-40 is fully cognate; 12/40 contains 12 cognate nucleotides centered around the restriction site and non-cognate ends, Table 1 ); gDNA, presence or absence of mouse genomic DNA (80 ng per reaction); Wash, whether the beads carrying immobilized Ramp conjugates were washed after target hybridization prior to addition of the Rrec BglII. The Y-axis shows the background-subtracted HRP signal values (with the background calculated as the mean signal generated for zero target concentrations). For normalization and comparison of sample series, the HRP signal values were expressed as the percentages of the maximum background-subtracted OD 655 obtained for the fully cognate target (the left-most bar of the graph). For all test samples, the target concentrations were 1 pM.

    Techniques Used: Sequencing, Hybridization, Generated

    14) Product Images from "Use of double-stranded DNA mini-circles to characterize the covalent topoisomerase-DNA complex"

    Article Title: Use of double-stranded DNA mini-circles to characterize the covalent topoisomerase-DNA complex

    Journal: Scientific Reports

    doi: 10.1038/srep13154

    Multiple Ct TopoI can be covalently linked to the dsMCs in the presence of camptothecin. ( a ) The relaxation of a mixture of the topoisomers T −4 and T −5 by the Ct TopoI (18.75 mU μL −1 (≈0.2 nM)) was performed in the absence (lane 2) or in the presence of 10 μM of camptothecin (CPT, lanes 3 and 4). After 10 minutes of incubation at 37 °C, SDS was added to the reaction mixtures to irreversibly trap the Ct TopoI covalently bound to DNA, and the samples were next treated with proteinase K. In lane 4, NaCl was added to the sample prior to SDS and proteinase K treatments. Samples were precipitated and analyzed on a 12% sequencing gel. dsMCs were digested with BamHI and HindIII (lane 1) or with BamHI and BglII (lane 5) and sequenced to determine the positions of the guanines and adenines in the sequence 36 . The size of single-stranded DNA fragments is indicated on the right side of the gel. lin., linear. circ., circular; ( b ) As in ( a ) except for the concentrations of camptothecin (CPT; 0 (lanes 3 and 4); 0.05 (lanes 5 and 6); 0.1 (lanes 7 and 8); 0.25 (lanes 9 and 10); 0.5 (lanes 11 and 12); 1 (lanes 13 and 14); 5 (lanes 15 and 16); 10 μM (lanes 17 and 18)) and the gel analysis (1% agarose gel, native conditions). When indicated, the samples were next treated with proteinase K (Prot K). Protein-free DNA and covalent Ct TopoI-DNA complexes are indicated. Lanes 1 and 2 contained 4% DMSO (no Ct TopoI no camptothecin); ( c ) As in ( a ) except for the concentrations of camptothecin (CPT; 0 (lane 3); 0.1 (lane 4); 0.25 (lane 5); 0.5 (lane 6); 1 (lane 7); 10 μM (lane 8)). dsMCs were digested with BamHI and HindIII (lane 1) or BamHI and BglII (lane 9) and sequenced to determine the positions of the guanines and adenines in the sequence 36 . Lane 2, mixture of T −4 and T −5 . The size of single-stranded DNA fragments is indicated on the right side of the gel.
    Figure Legend Snippet: Multiple Ct TopoI can be covalently linked to the dsMCs in the presence of camptothecin. ( a ) The relaxation of a mixture of the topoisomers T −4 and T −5 by the Ct TopoI (18.75 mU μL −1 (≈0.2 nM)) was performed in the absence (lane 2) or in the presence of 10 μM of camptothecin (CPT, lanes 3 and 4). After 10 minutes of incubation at 37 °C, SDS was added to the reaction mixtures to irreversibly trap the Ct TopoI covalently bound to DNA, and the samples were next treated with proteinase K. In lane 4, NaCl was added to the sample prior to SDS and proteinase K treatments. Samples were precipitated and analyzed on a 12% sequencing gel. dsMCs were digested with BamHI and HindIII (lane 1) or with BamHI and BglII (lane 5) and sequenced to determine the positions of the guanines and adenines in the sequence 36 . The size of single-stranded DNA fragments is indicated on the right side of the gel. lin., linear. circ., circular; ( b ) As in ( a ) except for the concentrations of camptothecin (CPT; 0 (lanes 3 and 4); 0.05 (lanes 5 and 6); 0.1 (lanes 7 and 8); 0.25 (lanes 9 and 10); 0.5 (lanes 11 and 12); 1 (lanes 13 and 14); 5 (lanes 15 and 16); 10 μM (lanes 17 and 18)) and the gel analysis (1% agarose gel, native conditions). When indicated, the samples were next treated with proteinase K (Prot K). Protein-free DNA and covalent Ct TopoI-DNA complexes are indicated. Lanes 1 and 2 contained 4% DMSO (no Ct TopoI no camptothecin); ( c ) As in ( a ) except for the concentrations of camptothecin (CPT; 0 (lane 3); 0.1 (lane 4); 0.25 (lane 5); 0.5 (lane 6); 1 (lane 7); 10 μM (lane 8)). dsMCs were digested with BamHI and HindIII (lane 1) or BamHI and BglII (lane 9) and sequenced to determine the positions of the guanines and adenines in the sequence 36 . Lane 2, mixture of T −4 and T −5 . The size of single-stranded DNA fragments is indicated on the right side of the gel.

    Techniques Used: Cycling Probe Technology, Incubation, Sequencing, Agarose Gel Electrophoresis

    Determination of the sequence of the camptothecin-induced cleavage sites. ( a ) Identification of the camptothecin-induced cleavage sites on the BamHI-digested dsMCs. The dsMC linearized by BamHI was treated with Ct TopoI (18.75 mU μL −1 (≈0.2 nM)) in the presence of 10 μM camptothecin as described in the methods section. After 10 minutes of incubation at 37 °C SDS was added to irreversibly trap the Ct TopoI covalently bound to DNA. The sample was next treated with proteinase K and precipitated. Lanes 5 and 10, sample directly loaded on a sequencing gel after precipitation. Sample further digested with BglII (lanes 6 and 9) or HindIII (lanes 7 and 8). dsMCs were digested with BamHI and BglII (lanes 1, 2 and 12) or BamHI and HindIII (lanes 3, 4 and 11) and sequenced to determine the positions of the guanines (lanes 1, 3 and 11) or the guanines and adenines (lanes 2, 4 and 12) in the sequence 36 . Samples loaded onto a 10% (lanes 1–7) or 7% (lanes 8–12) acrylamide sequencing gel. The camptothecin-induced cleavage sites are labelled from S 1 to S 14 ; ( b ) Overlayed scans of lanes 8 and 9 from site S 1 to S 8 . Black line, scan of lane 8 (BamHI-BglII double digestion). Blue line, scan of lane 9 (BamHI-HindIII double digestion); a. u., arbitrary unit; ( c ) The linearized dsMCs were incubated with Ct TopoI (18.75 mU μL −1 (≈0.2 nM)) and increasing concentrations of camptothecin [CPT; 0.05 (lanes 2 and 11); 0.1 (lanes 3 and 12); 0.2 (lanes 4 and 13); 0.5 (lanes 5 and 14); 1 (lanes 6 and 15); 5 (lanes 7 and 16); 10 μM (lanes 8 and 17)] as described in the methods section. After 10 minutes of incubation at 37 °C, SDS was added to the reaction mixtures to irreversibly trap the Ct TopoI covalently bound to DNA. When indicated, the samples were next treated with proteinase K (Prot K). The products of the reactions were finally analyzed on a 1% agarose gel under native conditions. Lane 9 contained 4% DMSO (no Ct TopoI no camptothecin). Lanes 1 and 10 contained 4% DMSO instead of camptothecin.
    Figure Legend Snippet: Determination of the sequence of the camptothecin-induced cleavage sites. ( a ) Identification of the camptothecin-induced cleavage sites on the BamHI-digested dsMCs. The dsMC linearized by BamHI was treated with Ct TopoI (18.75 mU μL −1 (≈0.2 nM)) in the presence of 10 μM camptothecin as described in the methods section. After 10 minutes of incubation at 37 °C SDS was added to irreversibly trap the Ct TopoI covalently bound to DNA. The sample was next treated with proteinase K and precipitated. Lanes 5 and 10, sample directly loaded on a sequencing gel after precipitation. Sample further digested with BglII (lanes 6 and 9) or HindIII (lanes 7 and 8). dsMCs were digested with BamHI and BglII (lanes 1, 2 and 12) or BamHI and HindIII (lanes 3, 4 and 11) and sequenced to determine the positions of the guanines (lanes 1, 3 and 11) or the guanines and adenines (lanes 2, 4 and 12) in the sequence 36 . Samples loaded onto a 10% (lanes 1–7) or 7% (lanes 8–12) acrylamide sequencing gel. The camptothecin-induced cleavage sites are labelled from S 1 to S 14 ; ( b ) Overlayed scans of lanes 8 and 9 from site S 1 to S 8 . Black line, scan of lane 8 (BamHI-BglII double digestion). Blue line, scan of lane 9 (BamHI-HindIII double digestion); a. u., arbitrary unit; ( c ) The linearized dsMCs were incubated with Ct TopoI (18.75 mU μL −1 (≈0.2 nM)) and increasing concentrations of camptothecin [CPT; 0.05 (lanes 2 and 11); 0.1 (lanes 3 and 12); 0.2 (lanes 4 and 13); 0.5 (lanes 5 and 14); 1 (lanes 6 and 15); 5 (lanes 7 and 16); 10 μM (lanes 8 and 17)] as described in the methods section. After 10 minutes of incubation at 37 °C, SDS was added to the reaction mixtures to irreversibly trap the Ct TopoI covalently bound to DNA. When indicated, the samples were next treated with proteinase K (Prot K). The products of the reactions were finally analyzed on a 1% agarose gel under native conditions. Lane 9 contained 4% DMSO (no Ct TopoI no camptothecin). Lanes 1 and 10 contained 4% DMSO instead of camptothecin.

    Techniques Used: Sequencing, Incubation, Cycling Probe Technology, Agarose Gel Electrophoresis

    15) Product Images from "Rejoining of DNA Double-Strand Breaks as a Function of Overhang Length †"

    Article Title: Rejoining of DNA Double-Strand Breaks as a Function of Overhang Length †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.25.3.896-906.2005

    Oligonucleotide-modified plasmid assay. (A) Plasmid modification scheme. pTW423 is digested with BglII and XhoI and purified, removing the polyterminator. Annealed oligonucleotides are then ligated onto the BglII and XhoI ends, restoring the ADE2 coding sequence. Precise in-frame repair of the break yields Ade + colonies. (B) Primer extension assay to determine the oligonucleotide ligation efficiency. Annealed oligonucleotides (Oligos) were added to the ligation reaction mixture at concentrations of 50, 100, 500, 1,000, and 5,000-fold molar excess over the concentration of the linearized plasmid (indicated by the thickness of the triangle over the lanes). Primer extension was performed after ligation as described in Materials and Methods.
    Figure Legend Snippet: Oligonucleotide-modified plasmid assay. (A) Plasmid modification scheme. pTW423 is digested with BglII and XhoI and purified, removing the polyterminator. Annealed oligonucleotides are then ligated onto the BglII and XhoI ends, restoring the ADE2 coding sequence. Precise in-frame repair of the break yields Ade + colonies. (B) Primer extension assay to determine the oligonucleotide ligation efficiency. Annealed oligonucleotides (Oligos) were added to the ligation reaction mixture at concentrations of 50, 100, 500, 1,000, and 5,000-fold molar excess over the concentration of the linearized plasmid (indicated by the thickness of the triangle over the lanes). Primer extension was performed after ligation as described in Materials and Methods.

    Techniques Used: Modification, Plasmid Preparation, Purification, Sequencing, Primer Extension Assay, Ligation, Concentration Assay

    16) 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

    17) Product Images from "Meiotic Transmission of an In Vitro–Assembled Autonomous Maize Minichromosome"

    Article Title: Meiotic Transmission of an In Vitro–Assembled Autonomous Maize Minichromosome

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0030179

    MMC Gene Expression and Structure (A) Fluorescent detection of nuclear-localized DsRed in MMC1 maize leaf; size bar, 50 μm. (B, C) Detection of DsRed sectors in a T2 plant leaf from event V-1 under (B) bright-field and (C) fluorescence microscopy; size bars, 0.5 mm. (D) high magnification view of image shown in (C) with the corresponding sector, comprising all cell layers, indicated by an asterisk; the edge of a sector that comprises only the adaxial cell layer is indicated by arrowheads, cells with typical DsRed expression are indicated by arrows. Size bar, 50 μm. (E) MMC consisting of a pCHR758 backbone and a centromere-derived insert, gene expression cassettes (grey), centromeric inserts (box), BglII restriction sites (arrowheads), and probes used for FISH and Southern blot analyses are indicated. (F) Southern blot of DNA digested with BglII and hybridized to probes 1–6 (E); Bands 1–4 measure 2,067, 3,167, 5,227 and 790 bp, respectively; those hybridizing to probes 5 and 6 vary in size, depending on the location of BglII sites within the centromeric DNA insert. MMC1 Control (c, lanes 1 and 5) DNA was purified from E. coli and hybridization patterns were compared to DNA from plant cell extracts derived from MMC1 events V-1 (lanes 2–4), Q-2 (lane 6), and V-4 (lane 7), as well as from plants transformed with pCHR758 (lane 8) and untransformed wild type (H99, lane 9). For events V-1 and Q-2, bands differing from bacterial grown controls are indicated (arrows and asterisk, respectively).
    Figure Legend Snippet: MMC Gene Expression and Structure (A) Fluorescent detection of nuclear-localized DsRed in MMC1 maize leaf; size bar, 50 μm. (B, C) Detection of DsRed sectors in a T2 plant leaf from event V-1 under (B) bright-field and (C) fluorescence microscopy; size bars, 0.5 mm. (D) high magnification view of image shown in (C) with the corresponding sector, comprising all cell layers, indicated by an asterisk; the edge of a sector that comprises only the adaxial cell layer is indicated by arrowheads, cells with typical DsRed expression are indicated by arrows. Size bar, 50 μm. (E) MMC consisting of a pCHR758 backbone and a centromere-derived insert, gene expression cassettes (grey), centromeric inserts (box), BglII restriction sites (arrowheads), and probes used for FISH and Southern blot analyses are indicated. (F) Southern blot of DNA digested with BglII and hybridized to probes 1–6 (E); Bands 1–4 measure 2,067, 3,167, 5,227 and 790 bp, respectively; those hybridizing to probes 5 and 6 vary in size, depending on the location of BglII sites within the centromeric DNA insert. MMC1 Control (c, lanes 1 and 5) DNA was purified from E. coli and hybridization patterns were compared to DNA from plant cell extracts derived from MMC1 events V-1 (lanes 2–4), Q-2 (lane 6), and V-4 (lane 7), as well as from plants transformed with pCHR758 (lane 8) and untransformed wild type (H99, lane 9). For events V-1 and Q-2, bands differing from bacterial grown controls are indicated (arrows and asterisk, respectively).

    Techniques Used: Expressing, Fluorescence, Microscopy, Derivative Assay, Fluorescence In Situ Hybridization, Southern Blot, Purification, Hybridization, Transformation Assay

    18) Product Images from "Enhancer Complexes Located Downstream of Both Human Immunoglobulin C? Genes "

    Article Title: Enhancer Complexes Located Downstream of Both Human Immunoglobulin C? Genes

    Journal: The Journal of Experimental Medicine

    doi:

    Mapping of DNase I hypersensitive sites in the regions 3′ of the human Cα genes. ( A ) DNase I hypersensitive sites lie downstream from the human Cα genes in the HS Sultan plasmacytoma. DNA samples prepared from DNase I–digested nuclei isolated from K562 promyeloid and HS Sultan myeloma cells were digested with BglII, electrophoresed, blotted, and hybridized with probe a (αm, Fig.1). No DNase I hypersensitive sites are seen in the K562 samples. In contrast, at least seven DNase I hypersensitive sites are observed in samples from HS Sultan plasmacytoma cells. The size of each DNase I–generated band corresponds to its distance from the BglII sites located ∼1 kb 5′ of each α membrane exon ( αm ). This mapping strategy does not distinguish between sites in the α1 versus α2 loci; sites are labeled according to their subsequent assignment (see B and C , and sequence analyses). Due to their large size, bands resulting from DNase I cutting at the α1 and α2 HS4 sites are not resolved in this analysis. ( B ) HS4 sites are accessible to nuclease in both α1 and α2 loci. HS Sultan nuclei were digested with DNase I or SspI restriction enzyme (both the α1 and α2 HS4 sequences contain an SspI site). Purified DNA was digested with EcoRI and hybridized with probe b′, yielding two closely spaced DNase I HS bands, whose sizes correspond to the expected distance between the HS4 enhancers and the downstream EcoRI sites. Furthermore, there are two similarly positioned bands in the samples from SspI-digested nuclei, indicating that both the α1 and α2 HS4 sites are accessible to SspI. ( C ) Assignment of DNase I hypersensitive sites to the 3′ Cα2 region. HS Sultan DNA samples were digested with HindIII and hybridized with probe g (α2 HS12, Fig. 1 ). Because DNAse I–generated bands from the α1 region which hybridize to this probe are expected to be larger than the 12-kb α2 HindIII fragment, all bands
    Figure Legend Snippet: Mapping of DNase I hypersensitive sites in the regions 3′ of the human Cα genes. ( A ) DNase I hypersensitive sites lie downstream from the human Cα genes in the HS Sultan plasmacytoma. DNA samples prepared from DNase I–digested nuclei isolated from K562 promyeloid and HS Sultan myeloma cells were digested with BglII, electrophoresed, blotted, and hybridized with probe a (αm, Fig.1). No DNase I hypersensitive sites are seen in the K562 samples. In contrast, at least seven DNase I hypersensitive sites are observed in samples from HS Sultan plasmacytoma cells. The size of each DNase I–generated band corresponds to its distance from the BglII sites located ∼1 kb 5′ of each α membrane exon ( αm ). This mapping strategy does not distinguish between sites in the α1 versus α2 loci; sites are labeled according to their subsequent assignment (see B and C , and sequence analyses). Due to their large size, bands resulting from DNase I cutting at the α1 and α2 HS4 sites are not resolved in this analysis. ( B ) HS4 sites are accessible to nuclease in both α1 and α2 loci. HS Sultan nuclei were digested with DNase I or SspI restriction enzyme (both the α1 and α2 HS4 sequences contain an SspI site). Purified DNA was digested with EcoRI and hybridized with probe b′, yielding two closely spaced DNase I HS bands, whose sizes correspond to the expected distance between the HS4 enhancers and the downstream EcoRI sites. Furthermore, there are two similarly positioned bands in the samples from SspI-digested nuclei, indicating that both the α1 and α2 HS4 sites are accessible to SspI. ( C ) Assignment of DNase I hypersensitive sites to the 3′ Cα2 region. HS Sultan DNA samples were digested with HindIII and hybridized with probe g (α2 HS12, Fig. 1 ). Because DNAse I–generated bands from the α1 region which hybridize to this probe are expected to be larger than the 12-kb α2 HindIII fragment, all bands

    Techniques Used: Isolation, Generated, Labeling, Sequencing, Purification

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    Southern Blot:

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

    Article Title: The molecular basis for stability of heterochromatin-mediated silencing in mammals
    Article Snippet: .. For methylation-sensitive restriction digest, DNA was digested with Bgl II and Hha I (New England Biolab). .. Completion of genomic DNA digestion was controlled with λ DNA (or a plasmid) digestion mixed with each reaction mix.

    Ligation:

    Article Title: Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium
    Article Snippet: .. The PCR fragment and plasmid pNZ8048 were both digested with restriction enzymes BglII and NcoI (New England BioLabs) and ligated at 16°C for 16 h. The resulting ligation was then transformed into electrocompetent E. coli MC1061 F′ (Lucigen). .. Successful transformants were identified by colony PCR using the primers pNZ8048-F (5′-GCCCCGTTAGTTGAAGAAGG-3′) and pNZ8048-R (5′-CAATTGAACGTTTCAAGCCTTGG-3′) and further verified by sequencing analysis.

    Construct:

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    Incubation:

    Article Title: Sp1 Regulates Chromatin Looping between an Intronic Enhancer and Distal Promoter of the Human Heme Oxygenase-1 Gene in Renal Cells *
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    other:

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

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    Polymerase Chain Reaction:

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    Transformation Assay:

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    New England Biolabs bglii
    Replication kinetics in Huh7.5 cells of the parental JFH1 and Jc1 full-length RNA transcripts and comparison with that of Jc1-BB containing introduced <t>BstBI</t> and <t>BglII</t> restriction sites in NS3 for protease gene insertion. The replication-defective mutant JFH1-GND was used as a negative control. The y axis records the percentage of HCV NS5A-positive cells scored by fluorescence microscopy.
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    Replication kinetics in Huh7.5 cells of the parental JFH1 and Jc1 full-length RNA transcripts and comparison with that of Jc1-BB containing introduced BstBI and BglII restriction sites in NS3 for protease gene insertion. The replication-defective mutant JFH1-GND was used as a negative control. The y axis records the percentage of HCV NS5A-positive cells scored by fluorescence microscopy.

    Journal: Journal of Virology

    Article Title: Development of an Intergenotypic Hepatitis C Virus (HCV) Cell Culture Method To Assess Antiviral Susceptibilities and Resistance Development of HCV NS3 Protease Genes from HCV Genotypes 1 to 6 ▿

    doi: 10.1128/JVI.02698-09

    Figure Lengend Snippet: Replication kinetics in Huh7.5 cells of the parental JFH1 and Jc1 full-length RNA transcripts and comparison with that of Jc1-BB containing introduced BstBI and BglII restriction sites in NS3 for protease gene insertion. The replication-defective mutant JFH1-GND was used as a negative control. The y axis records the percentage of HCV NS5A-positive cells scored by fluorescence microscopy.

    Article Snippet: Products were digested with BstBI and BglII (all restriction enzymes were obtained from New England Biolabs), gel purified (gel extraction kit; Qiagen) and ligated into pJFH1. pJFH1 has similarly been digested with BstBI and BglII, dephosphorylated using alkaline phosphatase (New England Biolabs), and gel purified.

    Techniques: Mutagenesis, Negative Control, Fluorescence, Microscopy

    Confirmation of chromatin looping by 3C assay with BglII and/or ApaLI restriction enzyme. A , schematic showing potential interaction between the fragments containing HS-2 region and the internal enhancer ( square ) and transcription factor(s) ( TF ). B , BglII

    Journal: The Journal of Biological Chemistry

    Article Title: Sp1 Regulates Chromatin Looping between an Intronic Enhancer and Distal Promoter of the Human Heme Oxygenase-1 Gene in Renal Cells *

    doi: 10.1074/jbc.M109.058586

    Figure Lengend Snippet: Confirmation of chromatin looping by 3C assay with BglII and/or ApaLI restriction enzyme. A , schematic showing potential interaction between the fragments containing HS-2 region and the internal enhancer ( square ) and transcription factor(s) ( TF ). B , BglII

    Article Snippet: SDS was then sequestered from the samples by the addition of 1.8% Triton X-100 (Fisher), and the samples were incubated at 37 °C for 1 h. The samples were digested with BglII and/or ApaLI restriction enzyme (New England Biolabs) at 37 °C for 16 h. Restriction enzymes were inactivated by the addition of 1.6% SDS and further incubation at 65 °C for 20 min.

    Techniques:

    Variability of Cre Expression of Mouse Line Tg Cre4 (A) Variable Cre expression in forebrains of three different mice positive for Tg Cre4 and R26R Cre indicator (see Figure S1 ) at postnatal day 12 pictured by the Cre-dependent β-galactosidase activity (blue, X-gal, counterstain by eosin) in coronal brain slices. Scale bar: 1.25 mm. (B) Southern blot analysis of BglII-digested genomic mouse DNA of four Tg Cre4 mice that differed in the Cre expression pattern. Southern probe detects the wild-type (4.5 kbp) and the transgenic (7, 5, 3, and 2 kbp) alleles.

    Journal: PLoS Biology

    Article Title: Enhanced Odor Discrimination and Impaired Olfactory Memory by Spatially Controlled Switch of AMPA ReceptorsGenetic and Behavioral Investigations into Odor Discrimination and Memory

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    Figure Lengend Snippet: Variability of Cre Expression of Mouse Line Tg Cre4 (A) Variable Cre expression in forebrains of three different mice positive for Tg Cre4 and R26R Cre indicator (see Figure S1 ) at postnatal day 12 pictured by the Cre-dependent β-galactosidase activity (blue, X-gal, counterstain by eosin) in coronal brain slices. Scale bar: 1.25 mm. (B) Southern blot analysis of BglII-digested genomic mouse DNA of four Tg Cre4 mice that differed in the Cre expression pattern. Southern probe detects the wild-type (4.5 kbp) and the transgenic (7, 5, 3, and 2 kbp) alleles.

    Article Snippet: Southern blot analysis Genomic DNA from mouse-tail/liver was digested with restriction enzyme BglII (NEB), and the Southern blot was done with a 320-bp probe (“integ”) obtained by PCR detecting the αCaMKII promoter.

    Techniques: Expressing, Mouse Assay, Activity Assay, Southern Blot, Transgenic Assay

    Maps of pNZC and inserts used in this study. A chloride-inducible promoter (CIP) was inserted between BglII and NcoI. P gadR is a constitutive promoter controlling the production of the activator protein GadR. lacZ and AMP expression are controlled by the chloride-inducible promoter P gad (activated by GadR). lacZ and Bac are inserted between cut sites NcoI and SpeI in pNZC to create pNZCL and pNZCA3.

    Journal: Applied and Environmental Microbiology

    Article Title: Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium

    doi: 10.1128/AEM.00227-15

    Figure Lengend Snippet: Maps of pNZC and inserts used in this study. A chloride-inducible promoter (CIP) was inserted between BglII and NcoI. P gadR is a constitutive promoter controlling the production of the activator protein GadR. lacZ and AMP expression are controlled by the chloride-inducible promoter P gad (activated by GadR). lacZ and Bac are inserted between cut sites NcoI and SpeI in pNZC to create pNZCL and pNZCA3.

    Article Snippet: The PCR fragment and plasmid pNZ8048 were both digested with restriction enzymes BglII and NcoI (New England BioLabs) and ligated at 16°C for 16 h. The resulting ligation was then transformed into electrocompetent E. coli MC1061 F′ (Lucigen).

    Techniques: Expressing, BAC Assay