bal31 nuclease  (New England Biolabs)


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    Name:
    Nuclease BAL 31
    Description:
    Nuclease BAL 31 50 units
    Catalog Number:
    m0213s
    Price:
    68
    Size:
    50 units
    Category:
    Exonucleases
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    Structured Review

    New England Biolabs bal31 nuclease
    Nuclease BAL 31
    Nuclease BAL 31 50 units
    https://www.bioz.com/result/bal31 nuclease/product/New England Biolabs
    Average 96 stars, based on 1096 article reviews
    Price from $9.99 to $1999.99
    bal31 nuclease - by Bioz Stars, 2020-08
    96/100 stars

    Images

    1) Product Images from "Cooperative kinking at distant sites in mechanically stressed DNA"

    Article Title: Cooperative kinking at distant sites in mechanically stressed DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr666

    DNA minicircles with 100, 106 and 108 bp, their purity and their susceptibility to Bal31 digestion. In a 10% denaturing PAGE gel (7 M urea), the denatured linear DNA fragments used as a marker, migrate much quicker than circular, covalently closed DNA minicircles in which the two strands cannot separate in space. Notice the presence of only one topoisomer in each category of minicircles and the sensitivity of minicircles with 100 bp to Bal31 nuclease.
    Figure Legend Snippet: DNA minicircles with 100, 106 and 108 bp, their purity and their susceptibility to Bal31 digestion. In a 10% denaturing PAGE gel (7 M urea), the denatured linear DNA fragments used as a marker, migrate much quicker than circular, covalently closed DNA minicircles in which the two strands cannot separate in space. Notice the presence of only one topoisomer in each category of minicircles and the sensitivity of minicircles with 100 bp to Bal31 nuclease.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Marker

    2) Product Images from "Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles"

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp137

    The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.
    Figure Legend Snippet: The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Ligation, Migration

    Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).
    Figure Legend Snippet: Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).

    Techniques Used: Agarose Gel Electrophoresis

    3) Product Images from "Production of DNA minicircles less than 250 base pairs through a novel concentrated DNA circularization assay enabling minicircle design with NF-κB inhibition activity"

    Article Title: Production of DNA minicircles less than 250 base pairs through a novel concentrated DNA circularization assay enabling minicircle design with NF-κB inhibition activity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1034

    CNDCA-based production of supercoiled minicircle. Image of stained native polyacrylamide gel showing 95 bp minicircle topoisomers of decreased linking number (ΔLk values of −1 and −2) (lanes 4, 6) as indicated on the right and obtained by religation of the nicked minicircle in the presence of EtBr. The slowest migrating band corresponds to the relaxed minicircle that migrates expectedly at the same rate as the nicked minicircle (lanes 1 and 2). Digestion of minicircle samples by the nuclease Bal31 is indicated on the right. The position of the molecular mass markers in bp is indicated on the left.
    Figure Legend Snippet: CNDCA-based production of supercoiled minicircle. Image of stained native polyacrylamide gel showing 95 bp minicircle topoisomers of decreased linking number (ΔLk values of −1 and −2) (lanes 4, 6) as indicated on the right and obtained by religation of the nicked minicircle in the presence of EtBr. The slowest migrating band corresponds to the relaxed minicircle that migrates expectedly at the same rate as the nicked minicircle (lanes 1 and 2). Digestion of minicircle samples by the nuclease Bal31 is indicated on the right. The position of the molecular mass markers in bp is indicated on the left.

    Techniques Used: Staining

    4) Product Images from "T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR"

    Article Title: T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

    Journal: Journal of Virology

    doi: 10.1128/JVI.01117-18

    T5 Exo efficiently removes rcDNA and genomic DNA from DNA preparation. (A) Copies (3 × 10 8 ) of virion DNA from purified HBV virions were incubated with PSD (5 U), T5 Exo (5 U), EcoRI (5 U), or DNase I (5 U) at 37°C for 1 h and further subjected to Southern blotting. pUCX3.2 plasmid (3.2 kb) was loaded as well to indicate the positions of rcDNA and cccDNA. (B) (Top) Two micrograms of purified 3.2-kb linear HBV monomer released from the pSHH2.1 plasmid by EcoRI digestion was incubated with indicated units of T5 Exo or PSD at 37°C for 1 h. (Middle) A mixture of 3.2-kb open circular DNA (2 μg) that was artificially nicked by Nb.BtsI endonuclease and 3.2-kb supercoiled pUCX3.2 plasmid (2 μg) was subjected to T5 Exo or PSD digestion at 37°C for 1 h. (Bottom) Two micrograms of genomic DNA from uninfected HepG2 hNTCP cells was similarly treated with T5 Exo or PSD. All digestion products are shown on agarose gels, and for relative quantification, band density of untreated samples is set as 100%. (C) Copies (10 8 ) of virion DNA or pUCX3.2 plasmid were digested with T5 Exo (5 U) or PSD (5 U) in the absence (0 μg) or presence (2 μg) of genomic DNA (as shown above; 1% agarose gel) at 37°C for 1 h, and the products were loaded for Southern blotting (bottom). (D) Virion DNA (rcV) or pSHH2.1 plasmid was incubated with T5 Exo (5 U) or PSD (10 U) at 37°C for 1 h, and products were further analyzed by pp466-541 (left) or pp1040-1996 (right), respectively. ns, no significance. (E) Total DNA samples from HBV-infected HepG2 hNTCP cells (days 1, 2, 3, 6, and 9 p.i. and day 0 without inocula) were incubated with T5 Exo (5 U) or PSD (10 U) as described above, and cccDNA (left) and total DNA (right) copies were quantified by respective primers.
    Figure Legend Snippet: T5 Exo efficiently removes rcDNA and genomic DNA from DNA preparation. (A) Copies (3 × 10 8 ) of virion DNA from purified HBV virions were incubated with PSD (5 U), T5 Exo (5 U), EcoRI (5 U), or DNase I (5 U) at 37°C for 1 h and further subjected to Southern blotting. pUCX3.2 plasmid (3.2 kb) was loaded as well to indicate the positions of rcDNA and cccDNA. (B) (Top) Two micrograms of purified 3.2-kb linear HBV monomer released from the pSHH2.1 plasmid by EcoRI digestion was incubated with indicated units of T5 Exo or PSD at 37°C for 1 h. (Middle) A mixture of 3.2-kb open circular DNA (2 μg) that was artificially nicked by Nb.BtsI endonuclease and 3.2-kb supercoiled pUCX3.2 plasmid (2 μg) was subjected to T5 Exo or PSD digestion at 37°C for 1 h. (Bottom) Two micrograms of genomic DNA from uninfected HepG2 hNTCP cells was similarly treated with T5 Exo or PSD. All digestion products are shown on agarose gels, and for relative quantification, band density of untreated samples is set as 100%. (C) Copies (10 8 ) of virion DNA or pUCX3.2 plasmid were digested with T5 Exo (5 U) or PSD (5 U) in the absence (0 μg) or presence (2 μg) of genomic DNA (as shown above; 1% agarose gel) at 37°C for 1 h, and the products were loaded for Southern blotting (bottom). (D) Virion DNA (rcV) or pSHH2.1 plasmid was incubated with T5 Exo (5 U) or PSD (10 U) at 37°C for 1 h, and products were further analyzed by pp466-541 (left) or pp1040-1996 (right), respectively. ns, no significance. (E) Total DNA samples from HBV-infected HepG2 hNTCP cells (days 1, 2, 3, 6, and 9 p.i. and day 0 without inocula) were incubated with T5 Exo (5 U) or PSD (10 U) as described above, and cccDNA (left) and total DNA (right) copies were quantified by respective primers.

    Techniques Used: Purification, Incubation, Southern Blot, Plasmid Preparation, Agarose Gel Electrophoresis, Infection

    5) Product Images from "Telomere length is highly heritable and independent of growth rate manipulated by temperature in field crickets"

    Article Title: Telomere length is highly heritable and independent of growth rate manipulated by temperature in field crickets

    Journal: bioRxiv

    doi: 10.1101/2020.05.29.123216

    Pulsed field gel electrophoresis showing TTAGG n telomere restriction fragments from the denatured chromosomes of six individual crickets (indicated by the numbers 1-6). XV molecular size ladders are shown on the outer lanes and shorter 1kb ladders are shown between crickets 2-3 and 4-5. The photo shows four aliquots of DNA sample per cricket and these aliquots were exposed to 0 (i.e. untreated), 20, 80, and 240 minutes of Bal 31 . Each time series clearly shows that Bal 31 decreases the molecular size distribution of TTAGG fragments. The absence of a banded distribution in the smears suggest that interstitial telomeric repeats were not present and that chromosome strands were intact. Taken together, these findings show that TTAGG n fragments were located at the chromosome-ends.
    Figure Legend Snippet: Pulsed field gel electrophoresis showing TTAGG n telomere restriction fragments from the denatured chromosomes of six individual crickets (indicated by the numbers 1-6). XV molecular size ladders are shown on the outer lanes and shorter 1kb ladders are shown between crickets 2-3 and 4-5. The photo shows four aliquots of DNA sample per cricket and these aliquots were exposed to 0 (i.e. untreated), 20, 80, and 240 minutes of Bal 31 . Each time series clearly shows that Bal 31 decreases the molecular size distribution of TTAGG fragments. The absence of a banded distribution in the smears suggest that interstitial telomeric repeats were not present and that chromosome strands were intact. Taken together, these findings show that TTAGG n fragments were located at the chromosome-ends.

    Techniques Used: Pulsed-Field Gel, Electrophoresis

    6) Product Images from "Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles"

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp137

    The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.
    Figure Legend Snippet: The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Ligation, Migration

    Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).
    Figure Legend Snippet: Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).

    Techniques Used: Agarose Gel Electrophoresis

    7) Product Images from "Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles"

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp137

    The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.
    Figure Legend Snippet: The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Ligation, Migration

    Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).
    Figure Legend Snippet: Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).

    Techniques Used: Agarose Gel Electrophoresis

    8) Product Images from "Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles"

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp137

    The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.
    Figure Legend Snippet: The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Ligation, Migration

    Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).
    Figure Legend Snippet: Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).

    Techniques Used: Agarose Gel Electrophoresis

    9) Product Images from "The mitochondrial genome of the pathogenic yeast Candida subhashii: GC-rich linear DNA with a protein covalently attached to the 5? termini"

    Article Title: The mitochondrial genome of the pathogenic yeast Candida subhashii: GC-rich linear DNA with a protein covalently attached to the 5? termini

    Journal: Microbiology

    doi: 10.1099/mic.0.038646-0

    The termini of the linear mtDNA are bound by a protein. (a) DNA samples were prepared from C. subhashii (see Methods) and separated by PFGE in a 1.5 % agarose gel. The gel was stained with 0.5 μg ml −1 ethidium bromide (EtBr) and transferred onto a nylon membrane. The blot was hybridized with radioactively labelled mtDNA from C. subhashii . Lane 1, isolated mtDNA; lanes 2 and 3, total cellular DNA prepared in agarose blocks treated or untreated with proteinase K, respectively. (b) Approximately 1 μg of isolated mtDNA was treated with exonuclease III (ExoIII) (left panel) or BAL-31 nuclease (right panel), as indicated. The mtDNA was then extracted from reactions, digested with Xba I endonuclease and electrophoretically separated. Note that the terminal fragments are sensitive to ExoIII but apparently not to BAL-31, indicating the possibility that the linear molecules have their 5′ termini blocked. L and R, positions of the 1527 and 2803 bp terminal restriction enzyme fragments, respectively; C, position of the internal control (a 1040 bp long linear blunt-ended DNA fragment) mixed with mtDNA prior to digestion with BAL-31 nuclease. (c) The mtDNA–protein complexes were isolated as described in Methods, digested with restriction endonucleases Cla I or Pvu I, and treated or not treated with proteinase K. The positions of terminal restriction enzyme fragments generated by Pvu I (833 and 2339 bp) and Cla I (547 bp) are indicated as L, R and L+R, respectively. Note that both terminal Cla I fragments have identical sizes, as this enzyme digests the C. subhashii mtDNA within TIRs.
    Figure Legend Snippet: The termini of the linear mtDNA are bound by a protein. (a) DNA samples were prepared from C. subhashii (see Methods) and separated by PFGE in a 1.5 % agarose gel. The gel was stained with 0.5 μg ml −1 ethidium bromide (EtBr) and transferred onto a nylon membrane. The blot was hybridized with radioactively labelled mtDNA from C. subhashii . Lane 1, isolated mtDNA; lanes 2 and 3, total cellular DNA prepared in agarose blocks treated or untreated with proteinase K, respectively. (b) Approximately 1 μg of isolated mtDNA was treated with exonuclease III (ExoIII) (left panel) or BAL-31 nuclease (right panel), as indicated. The mtDNA was then extracted from reactions, digested with Xba I endonuclease and electrophoretically separated. Note that the terminal fragments are sensitive to ExoIII but apparently not to BAL-31, indicating the possibility that the linear molecules have their 5′ termini blocked. L and R, positions of the 1527 and 2803 bp terminal restriction enzyme fragments, respectively; C, position of the internal control (a 1040 bp long linear blunt-ended DNA fragment) mixed with mtDNA prior to digestion with BAL-31 nuclease. (c) The mtDNA–protein complexes were isolated as described in Methods, digested with restriction endonucleases Cla I or Pvu I, and treated or not treated with proteinase K. The positions of terminal restriction enzyme fragments generated by Pvu I (833 and 2339 bp) and Cla I (547 bp) are indicated as L, R and L+R, respectively. Note that both terminal Cla I fragments have identical sizes, as this enzyme digests the C. subhashii mtDNA within TIRs.

    Techniques Used: Agarose Gel Electrophoresis, Staining, Isolation, Generated

    10) Product Images from "Telomere length is highly heritable and independent of growth rate manipulated by temperature in field crickets"

    Article Title: Telomere length is highly heritable and independent of growth rate manipulated by temperature in field crickets

    Journal: bioRxiv

    doi: 10.1101/2020.05.29.123216

    Pulsed field gel electrophoresis showing TTAGG n  telomere restriction fragments from the denatured chromosomes of six individual crickets (indicated by the numbers 1-6). XV molecular size ladders are shown on the outer lanes and shorter 1kb ladders are shown between crickets 2-3 and 4-5. The photo shows four aliquots of DNA sample per cricket and these aliquots were exposed to 0 (i.e. untreated), 20, 80, and 240 minutes of  Bal 31 . Each time series clearly shows that  Bal 31  decreases the molecular size distribution of TTAGG fragments. The absence of a banded distribution in the smears suggest that interstitial telomeric repeats were not present and that chromosome strands were intact. Taken together, these findings show that TTAGG n  fragments were located at the chromosome-ends.
    Figure Legend Snippet: Pulsed field gel electrophoresis showing TTAGG n telomere restriction fragments from the denatured chromosomes of six individual crickets (indicated by the numbers 1-6). XV molecular size ladders are shown on the outer lanes and shorter 1kb ladders are shown between crickets 2-3 and 4-5. The photo shows four aliquots of DNA sample per cricket and these aliquots were exposed to 0 (i.e. untreated), 20, 80, and 240 minutes of Bal 31 . Each time series clearly shows that Bal 31 decreases the molecular size distribution of TTAGG fragments. The absence of a banded distribution in the smears suggest that interstitial telomeric repeats were not present and that chromosome strands were intact. Taken together, these findings show that TTAGG n fragments were located at the chromosome-ends.

    Techniques Used: Pulsed-Field Gel, Electrophoresis

    11) Product Images from "Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles"

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp137

    The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.
    Figure Legend Snippet: The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Ligation, Migration

    Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).
    Figure Legend Snippet: Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).

    Techniques Used: Agarose Gel Electrophoresis

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

    Article Title: Structural diversity of supercoiled DNA

    Journal: Nature Communications

    doi: 10.1038/ncomms9440

    Mapping Bal-31 cleavage. To determine whether Bal-31 cleavage occurs at multiple sites or at a preferred site, the Δ Lk =−6 topoisomer was cleaved with Bal-31 and various restriction enzymes. ( a ) Products were separated by agarose gel electrophoresis. Left, (lanes 1–5), control reactions, mc336 (approximately equal mixture of Δ Lk =−2 and Δ Lk =−3 topoisomers) with combinations of the various restriction enzymes (as indicated) to generate fragments of known DNA lengths. Right, (lanes 6–9), Δ Lk =−6 topoisomer cleaved first with Bal-31, followed by a restriction enzyme (as indicated). Mr 1 : 100 bp DNA ladder, Mr 2 : Low molecular weight DNA ladder. ( b ) Map of the minicircle sequence showing the positions of the restriction enzymes used, the estimated location of Bal-31 cleavage (with parentheses indicating the range), and the location of the observed base-pair breaking in MD simulation of the Δ Lk =−3 topoisomer.
    Figure Legend Snippet: Mapping Bal-31 cleavage. To determine whether Bal-31 cleavage occurs at multiple sites or at a preferred site, the Δ Lk =−6 topoisomer was cleaved with Bal-31 and various restriction enzymes. ( a ) Products were separated by agarose gel electrophoresis. Left, (lanes 1–5), control reactions, mc336 (approximately equal mixture of Δ Lk =−2 and Δ Lk =−3 topoisomers) with combinations of the various restriction enzymes (as indicated) to generate fragments of known DNA lengths. Right, (lanes 6–9), Δ Lk =−6 topoisomer cleaved first with Bal-31, followed by a restriction enzyme (as indicated). Mr 1 : 100 bp DNA ladder, Mr 2 : Low molecular weight DNA ladder. ( b ) Map of the minicircle sequence showing the positions of the restriction enzymes used, the estimated location of Bal-31 cleavage (with parentheses indicating the range), and the location of the observed base-pair breaking in MD simulation of the Δ Lk =−3 topoisomer.

    Techniques Used: Agarose Gel Electrophoresis, Molecular Weight, Sequencing

    Effect of supercoiling on DNA base accessibility. ( a ) Minicircle DNA incubated with nuclease Bal-31. Over time, samples were removed, quenched by the addition of stop buffer and the products analysed by polyacrylamide gel electrophoresis. Mr: 100 bp DNA ladder, L: linearized 336 bp DNA, N: nicked 336 bp minicircle. ( b ) Graphic representation of the data shown in ( a ) Fitted lines are for visualization purposes only. ( c ) MD simulation of the Δ Lk =−3 topoisomer in explicit solvent. Splayed bases were found at a sharp bend of a needle conformation. This may be a potential atomistic explanation for Bal-31 susceptibility of negatively supercoiled topoisomers.
    Figure Legend Snippet: Effect of supercoiling on DNA base accessibility. ( a ) Minicircle DNA incubated with nuclease Bal-31. Over time, samples were removed, quenched by the addition of stop buffer and the products analysed by polyacrylamide gel electrophoresis. Mr: 100 bp DNA ladder, L: linearized 336 bp DNA, N: nicked 336 bp minicircle. ( b ) Graphic representation of the data shown in ( a ) Fitted lines are for visualization purposes only. ( c ) MD simulation of the Δ Lk =−3 topoisomer in explicit solvent. Splayed bases were found at a sharp bend of a needle conformation. This may be a potential atomistic explanation for Bal-31 susceptibility of negatively supercoiled topoisomers.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    13) Product Images from "Evolution of linear chromosomes and multipartite genomes in yeast mitochondria"

    Article Title: Evolution of linear chromosomes and multipartite genomes in yeast mitochondria

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1345

    Multipartite linear-mapping genomes in C. labiduridarum and C. frijolesensis ( A ) PFGE separated samples of C. labiduridarum NRRL Y-27940 (lane 1) and C. frijolesensis NRRL Y-48060 (lane 2) were blotted onto a nylon membrane and hybridized with the radioactively labeled probes P-668 and H-1030 (regions hybridizing with both probes are shown as dashed lines). Presumed master (I) and two smaller chromosomes (II and III) are indicated. Note that the master chromosome occurs in four isomers (i.e. L III − R III − L II − R II (shown in the scheme), L III − R III − R II − L II , R III − L III − L II − R II and R III − L III − R II − L II . ‘L’ and ‘R’ indicate the left and the right telomere, respectively). The C. frijolesensis mtDNA (∼1 µg) was digested with BAL-31 nuclease ( B ) or exonuclease III (ExoIII) ( C ) as indicated. After nuclease inactivation, the DNA was digested with EcoRV, separated in 0.9% (w/v) agarose gel. The Southern blots were hybridized with the P-668 and EH-1350 probes specific for the left and the right arm of the master chromosome, respectively (see ‘Materials and Methods’ section). Arrows show the positions of the left (L) and right (R) terminal fragments and their fusions (R + R, R + L and L + L). Note that after ExoIII treatment the telomeric fragments form two subpopulations that differ in their sensitivity to the ExoIII treatment. This indicates that the linear mtDNA molecules possess an open structure with 5′ overhang or blunt end or covalently closed t-hairpin. ( D ) The C. frijolesensis mtDNA was treated with antarctic phosphatase and labeled with [γ 32 P]ATP and T4 polynucleotide kinase. The mtDNA was then digested with restriction endonuclease EcoRV (lane 1) or BglII (lane 2) and separated in 0.8% (w/v) agarose gel (left panel). The gel was fixed in 10% (v/v) methanol/10% (v/v) acetic acid for 30 min, dried overnight and autoradiographed (right panel). Arrows indicate the position of telomeric fragments containing the open structures accessible to terminal labeling.
    Figure Legend Snippet: Multipartite linear-mapping genomes in C. labiduridarum and C. frijolesensis ( A ) PFGE separated samples of C. labiduridarum NRRL Y-27940 (lane 1) and C. frijolesensis NRRL Y-48060 (lane 2) were blotted onto a nylon membrane and hybridized with the radioactively labeled probes P-668 and H-1030 (regions hybridizing with both probes are shown as dashed lines). Presumed master (I) and two smaller chromosomes (II and III) are indicated. Note that the master chromosome occurs in four isomers (i.e. L III − R III − L II − R II (shown in the scheme), L III − R III − R II − L II , R III − L III − L II − R II and R III − L III − R II − L II . ‘L’ and ‘R’ indicate the left and the right telomere, respectively). The C. frijolesensis mtDNA (∼1 µg) was digested with BAL-31 nuclease ( B ) or exonuclease III (ExoIII) ( C ) as indicated. After nuclease inactivation, the DNA was digested with EcoRV, separated in 0.9% (w/v) agarose gel. The Southern blots were hybridized with the P-668 and EH-1350 probes specific for the left and the right arm of the master chromosome, respectively (see ‘Materials and Methods’ section). Arrows show the positions of the left (L) and right (R) terminal fragments and their fusions (R + R, R + L and L + L). Note that after ExoIII treatment the telomeric fragments form two subpopulations that differ in their sensitivity to the ExoIII treatment. This indicates that the linear mtDNA molecules possess an open structure with 5′ overhang or blunt end or covalently closed t-hairpin. ( D ) The C. frijolesensis mtDNA was treated with antarctic phosphatase and labeled with [γ 32 P]ATP and T4 polynucleotide kinase. The mtDNA was then digested with restriction endonuclease EcoRV (lane 1) or BglII (lane 2) and separated in 0.8% (w/v) agarose gel (left panel). The gel was fixed in 10% (v/v) methanol/10% (v/v) acetic acid for 30 min, dried overnight and autoradiographed (right panel). Arrows indicate the position of telomeric fragments containing the open structures accessible to terminal labeling.

    Techniques Used: Labeling, Agarose Gel Electrophoresis

    Circular- and linear-mapping genome isomers in mitochondria of C. viswanathii. ( A ) The mtDNA samples were digested with BamHI (lane 1) or Eco91I (lane 2) and separated in 1% (w/v) agarose gel. The Southern blot was hybridized with radioactively labeled oligonucleotide probe Oligo-32 derived from the large palindrome (shown as dashed arrows). The solid arrows show positions of the palindrome and the presumed terminal fragments of resolved linear molecules capped with t-hairpins. Scheme shows presumed circular- (I) and linear-mapping (II) genome isomers. ( B ) Isolated mtDNA was treated or untreated with BAL-31 nuclease (0.2 U for 5 min). The mtDNA was then extracted from the reaction, digested with BamHI or Eco91I endonuclease, and electrophoretically separated. Note that the fragments containing presumed t-hairpins were sensitive to BAL-31 nuclease (indicated by asterisk).
    Figure Legend Snippet: Circular- and linear-mapping genome isomers in mitochondria of C. viswanathii. ( A ) The mtDNA samples were digested with BamHI (lane 1) or Eco91I (lane 2) and separated in 1% (w/v) agarose gel. The Southern blot was hybridized with radioactively labeled oligonucleotide probe Oligo-32 derived from the large palindrome (shown as dashed arrows). The solid arrows show positions of the palindrome and the presumed terminal fragments of resolved linear molecules capped with t-hairpins. Scheme shows presumed circular- (I) and linear-mapping (II) genome isomers. ( B ) Isolated mtDNA was treated or untreated with BAL-31 nuclease (0.2 U for 5 min). The mtDNA was then extracted from the reaction, digested with BamHI or Eco91I endonuclease, and electrophoretically separated. Note that the fragments containing presumed t-hairpins were sensitive to BAL-31 nuclease (indicated by asterisk).

    Techniques Used: Agarose Gel Electrophoresis, Southern Blot, Labeling, Derivative Assay, Isolation

    14) Product Images from "Cooperative kinking at distant sites in mechanically stressed DNA"

    Article Title: Cooperative kinking at distant sites in mechanically stressed DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr666

    DNA minicircles with 100, 106 and 108 bp, their purity and their susceptibility to Bal31 digestion. In a 10% denaturing PAGE gel (7 M urea), the denatured linear DNA fragments used as a marker, migrate much quicker than circular, covalently closed DNA minicircles in which the two strands cannot separate in space. Notice the presence of only one topoisomer in each category of minicircles and the sensitivity of minicircles with 100 bp to Bal31 nuclease.
    Figure Legend Snippet: DNA minicircles with 100, 106 and 108 bp, their purity and their susceptibility to Bal31 digestion. In a 10% denaturing PAGE gel (7 M urea), the denatured linear DNA fragments used as a marker, migrate much quicker than circular, covalently closed DNA minicircles in which the two strands cannot separate in space. Notice the presence of only one topoisomer in each category of minicircles and the sensitivity of minicircles with 100 bp to Bal31 nuclease.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Marker

    15) Product Images from "Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles"

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp137

    The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.
    Figure Legend Snippet: The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Ligation, Migration

    Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).
    Figure Legend Snippet: Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).

    Techniques Used: Agarose Gel Electrophoresis

    16) Product Images from "Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles"

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp137

    The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.
    Figure Legend Snippet: The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Ligation, Migration

    Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).
    Figure Legend Snippet: Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).

    Techniques Used: Agarose Gel Electrophoresis

    17) Product Images from "Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles"

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp137

    The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.
    Figure Legend Snippet: The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Ligation, Migration

    Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).
    Figure Legend Snippet: Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).

    Techniques Used: Agarose Gel Electrophoresis

    18) Product Images from "Production of DNA minicircles less than 250 base pairs through a novel concentrated DNA circularization assay enabling minicircle design with NF-κB inhibition activity"

    Article Title: Production of DNA minicircles less than 250 base pairs through a novel concentrated DNA circularization assay enabling minicircle design with NF-κB inhibition activity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1034

    CNDCA-based production of supercoiled minicircle. Image of stained native polyacrylamide gel showing 95 bp minicircle topoisomers of decreased linking number (ΔLk values of −1 and −2) (lanes 4, 6) as indicated on the right and obtained by religation of the nicked minicircle in the presence of EtBr. The slowest migrating band corresponds to the relaxed minicircle that migrates expectedly at the same rate as the nicked minicircle (lanes 1 and 2). Digestion of minicircle samples by the nuclease Bal31 is indicated on the right. The position of the molecular mass markers in bp is indicated on the left.
    Figure Legend Snippet: CNDCA-based production of supercoiled minicircle. Image of stained native polyacrylamide gel showing 95 bp minicircle topoisomers of decreased linking number (ΔLk values of −1 and −2) (lanes 4, 6) as indicated on the right and obtained by religation of the nicked minicircle in the presence of EtBr. The slowest migrating band corresponds to the relaxed minicircle that migrates expectedly at the same rate as the nicked minicircle (lanes 1 and 2). Digestion of minicircle samples by the nuclease Bal31 is indicated on the right. The position of the molecular mass markers in bp is indicated on the left.

    Techniques Used: Staining

    19) Product Images from "Brochothrix thermosphacta Bacteriophages Feature Heterogeneous and Highly Mosaic Genomes and Utilize Unique Prophage Insertion Sites ▿ Bacteriophages Feature Heterogeneous and Highly Mosaic Genomes and Utilize Unique Prophage Insertion Sites ▿ †"

    Article Title: Brochothrix thermosphacta Bacteriophages Feature Heterogeneous and Highly Mosaic Genomes and Utilize Unique Prophage Insertion Sites ▿ Bacteriophages Feature Heterogeneous and Highly Mosaic Genomes and Utilize Unique Prophage Insertion Sites ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00709-10

    Structure analysis of the DNA molecules of phage A9, NF5, and BL3. (A) PFGE of full-length phage DNA. Size markers: λmix, lambda mix marker 19 (Fermentas); M1 and M2, PFG midrange markers I and II (NEB). BL3 and NF5 genomic DNAs run at slightly larger sizes than their unit genome lengths of 41.3 and 37 kb. A9 DNA exhibits approximately the same physical size as Listeria ). (B) Gel electrophoresis of PacI-digested A9 DNA after Bal31 nuclease treatment for the indicated time intervals. Terminal fragments of the nonpermuted genomes disappear over time (indicated by arrows). (C) Restriction enzyme profiles of PstI-digested NF5 DNA and SwaI-digested BL3 DNA; the putative pac fragments are indicated by black arrowheads. (D and E) EcoRI (NF5)- and Van91I (BL3)-digested genomic DNA after Bal31 nuclease treatment for the indicated time intervals. Arrows with square heads indicate restriction fragments which were not predicted in silico from a circular molecule ( pac fragment, 4.2 kb; smeary band, > 10 kb). The “submolar” pac fragment disappears after 10 min of Bal31 treatment. Arrows with pointed heads indicate restriction fragments “downstream” from the putative pac site-containing fragment. Their consecutive appearance within the respective genome is indicated by numbering and correlates with the order of disappearance, indicating incomplete permutation of the DNA molecules based on the limited number of genomes in each replication concatemer.
    Figure Legend Snippet: Structure analysis of the DNA molecules of phage A9, NF5, and BL3. (A) PFGE of full-length phage DNA. Size markers: λmix, lambda mix marker 19 (Fermentas); M1 and M2, PFG midrange markers I and II (NEB). BL3 and NF5 genomic DNAs run at slightly larger sizes than their unit genome lengths of 41.3 and 37 kb. A9 DNA exhibits approximately the same physical size as Listeria ). (B) Gel electrophoresis of PacI-digested A9 DNA after Bal31 nuclease treatment for the indicated time intervals. Terminal fragments of the nonpermuted genomes disappear over time (indicated by arrows). (C) Restriction enzyme profiles of PstI-digested NF5 DNA and SwaI-digested BL3 DNA; the putative pac fragments are indicated by black arrowheads. (D and E) EcoRI (NF5)- and Van91I (BL3)-digested genomic DNA after Bal31 nuclease treatment for the indicated time intervals. Arrows with square heads indicate restriction fragments which were not predicted in silico from a circular molecule ( pac fragment, 4.2 kb; smeary band, > 10 kb). The “submolar” pac fragment disappears after 10 min of Bal31 treatment. Arrows with pointed heads indicate restriction fragments “downstream” from the putative pac site-containing fragment. Their consecutive appearance within the respective genome is indicated by numbering and correlates with the order of disappearance, indicating incomplete permutation of the DNA molecules based on the limited number of genomes in each replication concatemer.

    Techniques Used: Marker, Nucleic Acid Electrophoresis, In Silico

    20) Product Images from "Novel Virulent and Broad-Host-Range Erwinia amylovora Bacteriophages Reveal a High Degree of Mosaicism and a Relationship to Enterobacteriaceae Phages ▿ Phages ▿ †"

    Article Title: Novel Virulent and Broad-Host-Range Erwinia amylovora Bacteriophages Reveal a High Degree of Mosaicism and a Relationship to Enterobacteriaceae Phages ▿ Phages ▿ †

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.03022-10

    Analysis of phage genome physical structures. Shown are fragment patterns of phage DNA, following time-limited BAL31 treatment and subsequent digestion with different endonucleases. Phage L1 was digested with MfeI, M7 with FspBI, S6 with three different
    Figure Legend Snippet: Analysis of phage genome physical structures. Shown are fragment patterns of phage DNA, following time-limited BAL31 treatment and subsequent digestion with different endonucleases. Phage L1 was digested with MfeI, M7 with FspBI, S6 with three different

    Techniques Used:

    21) Product Images from "Dynamic Evolution of Telomeric Sequences in the Green Algal Order Chlamydomonadales"

    Article Title: Dynamic Evolution of Telomeric Sequences in the Green Algal Order Chlamydomonadales

    Journal: Genome Biology and Evolution

    doi: 10.1093/gbe/evs007

    BAL-31 nuclease treatment of intact genomic DNA from the Dunaliellinia (TEL168) and the Stephanosphaeria (TEL157, TEL180). High-molecular weight DNA samples after BAL-31 nuclease treatment (15 and 45 min) and restriction endonuclease (RE) digestion (−, non digested) were analyzed by pulse-field gel electrophoresis (marker lengths in kilobase). The hybridization pattern of the probes specific for the human-type (HUSB) and the Arabidopsis -type (ATSB) telomeric sequence revealed their terminal position by a reduction of signal intensity with increasing duration of BAL-31 digestion.
    Figure Legend Snippet: BAL-31 nuclease treatment of intact genomic DNA from the Dunaliellinia (TEL168) and the Stephanosphaeria (TEL157, TEL180). High-molecular weight DNA samples after BAL-31 nuclease treatment (15 and 45 min) and restriction endonuclease (RE) digestion (−, non digested) were analyzed by pulse-field gel electrophoresis (marker lengths in kilobase). The hybridization pattern of the probes specific for the human-type (HUSB) and the Arabidopsis -type (ATSB) telomeric sequence revealed their terminal position by a reduction of signal intensity with increasing duration of BAL-31 digestion.

    Techniques Used: Molecular Weight, Nucleic Acid Electrophoresis, Marker, Hybridization, Sequencing

    22) Product Images from "Cooperative kinking at distant sites in mechanically stressed DNA"

    Article Title: Cooperative kinking at distant sites in mechanically stressed DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr666

    DNA minicircles with 100, 106 and 108 bp, their purity and their susceptibility to Bal31 digestion. In a 10% denaturing PAGE gel (7 M urea), the denatured linear DNA fragments used as a marker, migrate much quicker than circular, covalently closed DNA minicircles in which the two strands cannot separate in space. Notice the presence of only one topoisomer in each category of minicircles and the sensitivity of minicircles with 100 bp to Bal31 nuclease.
    Figure Legend Snippet: DNA minicircles with 100, 106 and 108 bp, their purity and their susceptibility to Bal31 digestion. In a 10% denaturing PAGE gel (7 M urea), the denatured linear DNA fragments used as a marker, migrate much quicker than circular, covalently closed DNA minicircles in which the two strands cannot separate in space. Notice the presence of only one topoisomer in each category of minicircles and the sensitivity of minicircles with 100 bp to Bal31 nuclease.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Marker

    23) Product Images from "Cooperative kinking at distant sites in mechanically stressed DNA"

    Article Title: Cooperative kinking at distant sites in mechanically stressed DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr666

    DNA minicircles with 100, 106 and 108 bp, their purity and their susceptibility to Bal31 digestion. In a 10% denaturing PAGE gel (7 M urea), the denatured linear DNA fragments used as a marker, migrate much quicker than circular, covalently closed DNA minicircles in which the two strands cannot separate in space. Notice the presence of only one topoisomer in each category of minicircles and the sensitivity of minicircles with 100 bp to Bal31 nuclease.
    Figure Legend Snippet: DNA minicircles with 100, 106 and 108 bp, their purity and their susceptibility to Bal31 digestion. In a 10% denaturing PAGE gel (7 M urea), the denatured linear DNA fragments used as a marker, migrate much quicker than circular, covalently closed DNA minicircles in which the two strands cannot separate in space. Notice the presence of only one topoisomer in each category of minicircles and the sensitivity of minicircles with 100 bp to Bal31 nuclease.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Marker

    24) Product Images from "Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles"

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp137

    The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.
    Figure Legend Snippet: The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Ligation, Migration

    Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).
    Figure Legend Snippet: Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).

    Techniques Used: Agarose Gel Electrophoresis

    25) Product Images from "Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles"

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp137

    The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.
    Figure Legend Snippet: The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Ligation, Migration

    Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).
    Figure Legend Snippet: Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).

    Techniques Used: Agarose Gel Electrophoresis

    26) Product Images from "Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles"

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp137

    The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.
    Figure Legend Snippet: The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Ligation, Migration

    Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).
    Figure Legend Snippet: Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).

    Techniques Used: Agarose Gel Electrophoresis

    27) Product Images from "Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles"

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp137

    The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.
    Figure Legend Snippet: The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Ligation, Migration

    Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).
    Figure Legend Snippet: Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).

    Techniques Used: Agarose Gel Electrophoresis

    28) Product Images from "Chromatin features of plant telomeric sequences at terminal vs. internal positions"

    Article Title: Chromatin features of plant telomeric sequences at terminal vs. internal positions

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2014.00593

    Internal localization of telomeric repeats in the B. antipoda genome. (A) High molecular weight DNA digested by Bal31 nuclease and a mix of frequently cutting restriction enzymes was analyzed by pulse field gel electrophoresis. Signals after hybridization with the radioactively labeled telomeric oligonucleotide showed resistance of telomeric sequences to Bal31 cleavage. The apparent decrease in signal intensity after 30 min of Bal31 digestion reflects variation in sample loading rather than a change due to the Bal31 treatment (compare to the signal after 45 min of Bal31). (B) DNA released into solution in the course of Bal31 digestions was purified and subjected to conventional agarose electrophoresis. The numbers above lines indicate the time of Bal31 digestion in minutes. M, DNA size markers.
    Figure Legend Snippet: Internal localization of telomeric repeats in the B. antipoda genome. (A) High molecular weight DNA digested by Bal31 nuclease and a mix of frequently cutting restriction enzymes was analyzed by pulse field gel electrophoresis. Signals after hybridization with the radioactively labeled telomeric oligonucleotide showed resistance of telomeric sequences to Bal31 cleavage. The apparent decrease in signal intensity after 30 min of Bal31 digestion reflects variation in sample loading rather than a change due to the Bal31 treatment (compare to the signal after 45 min of Bal31). (B) DNA released into solution in the course of Bal31 digestions was purified and subjected to conventional agarose electrophoresis. The numbers above lines indicate the time of Bal31 digestion in minutes. M, DNA size markers.

    Techniques Used: Molecular Weight, Nucleic Acid Electrophoresis, Hybridization, Labeling, Purification, Electrophoresis

    Relative cytosine methylation in telomeres of Nicotiana species. (A) After Bal31 treatment of N. tabacum high molecular weight DNA, TRF analysis was used to control the efficiency of digestion. A loss of the telomere-specific hybridization signal was observed in the course of Bal31 treatment. After 15 min of Bal31 digestion loss of the signal is evident but telomere erosion is not clear, whereas after 45- and 90-min treatments, telomeres were efficiently degraded. Time of Bal31 digestion is given above the lanes. M – DNA size marker. (B) Dot-blot analysis of Bal31-digested DNA from N. tabacum and N. tomentosiformis after treatment with sodium bisulfite. Samples were loaded onto a membrane and hybridized with radioactively labeled probes to detect the total signal of telomeres (loading probe pltel-C complementary to the telomeric G-strand) and the portion of methylated telomeres (DEGENER probe). Time of the Bal31 digestion in minutes is given above the membranes. +, positive hybridization control (tobacco DNA without the bisulfite treatment); −, negative control (DNA from the pUC19 plasmid). (C) Relative density of methylated cytosines along telomeres, calculated as the DEGENER/loading hybridization signals ratio. The ratio in Bal31 non-treated samples was arbitrarily taken as 1. Six independent experiments were performed.
    Figure Legend Snippet: Relative cytosine methylation in telomeres of Nicotiana species. (A) After Bal31 treatment of N. tabacum high molecular weight DNA, TRF analysis was used to control the efficiency of digestion. A loss of the telomere-specific hybridization signal was observed in the course of Bal31 treatment. After 15 min of Bal31 digestion loss of the signal is evident but telomere erosion is not clear, whereas after 45- and 90-min treatments, telomeres were efficiently degraded. Time of Bal31 digestion is given above the lanes. M – DNA size marker. (B) Dot-blot analysis of Bal31-digested DNA from N. tabacum and N. tomentosiformis after treatment with sodium bisulfite. Samples were loaded onto a membrane and hybridized with radioactively labeled probes to detect the total signal of telomeres (loading probe pltel-C complementary to the telomeric G-strand) and the portion of methylated telomeres (DEGENER probe). Time of the Bal31 digestion in minutes is given above the membranes. +, positive hybridization control (tobacco DNA without the bisulfite treatment); −, negative control (DNA from the pUC19 plasmid). (C) Relative density of methylated cytosines along telomeres, calculated as the DEGENER/loading hybridization signals ratio. The ratio in Bal31 non-treated samples was arbitrarily taken as 1. Six independent experiments were performed.

    Techniques Used: Methylation, Molecular Weight, Hybridization, Marker, Dot Blot, Labeling, Negative Control, Plasmid Preparation

    Methylation of cytosines of B. antipoda telomeric repeats. (A) Analysis of global methylation of cytosines in telomeric repeats of B. antipoda . High molecular weight DNA was treated with Bal31, converted with bisulfite and analyzed by Southern hybridization against loading and DEGENER probes. Signals with the DEGENER probe indicating methylated cytosines in telomeric repeats were obtained in all samples. The time of Bal31 digestion is given above the membranes. +, positive hybridization control (tobacco DNA without the bisulfite treatment); −, negative control (DNA from pUC19 plasmid). (B) An example of bisulfite sequencing data for Ba493 and Ba576 regions in adult leaves. Non-symmetrically located cytosines are depicted as green triangles; full triangle, methylated; empty triangle, non-methylated cytosine. Cytosines located in perfect telomeric repeats are underlined. The first line of the scheme where all cytosines in all sequence contexts are presented as full figures represents position of respective cytosine. (C) Graphical representation of telomeric cytosine methylation within the ITR-containing sequences Ba493 and Ba576. Three tissues were analyzed to reveal possible tissue-specific methylation pattern. “all,” all cytosines located in non-symmetrical sequence context; “telomeric,” cytosines located in perfect telomeric repeats. Data were processed by One-Way independent ANOVA test with standard weighted-means, * is used for P
    Figure Legend Snippet: Methylation of cytosines of B. antipoda telomeric repeats. (A) Analysis of global methylation of cytosines in telomeric repeats of B. antipoda . High molecular weight DNA was treated with Bal31, converted with bisulfite and analyzed by Southern hybridization against loading and DEGENER probes. Signals with the DEGENER probe indicating methylated cytosines in telomeric repeats were obtained in all samples. The time of Bal31 digestion is given above the membranes. +, positive hybridization control (tobacco DNA without the bisulfite treatment); −, negative control (DNA from pUC19 plasmid). (B) An example of bisulfite sequencing data for Ba493 and Ba576 regions in adult leaves. Non-symmetrically located cytosines are depicted as green triangles; full triangle, methylated; empty triangle, non-methylated cytosine. Cytosines located in perfect telomeric repeats are underlined. The first line of the scheme where all cytosines in all sequence contexts are presented as full figures represents position of respective cytosine. (C) Graphical representation of telomeric cytosine methylation within the ITR-containing sequences Ba493 and Ba576. Three tissues were analyzed to reveal possible tissue-specific methylation pattern. “all,” all cytosines located in non-symmetrical sequence context; “telomeric,” cytosines located in perfect telomeric repeats. Data were processed by One-Way independent ANOVA test with standard weighted-means, * is used for P

    Techniques Used: Methylation, Molecular Weight, Hybridization, Negative Control, Plasmid Preparation, Methylation Sequencing, Sequencing

    29) Product Images from "T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR"

    Article Title: T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

    Journal: Journal of Virology

    doi: 10.1128/JVI.01117-18

    Identification of exonucleases selectively digesting rcDNA. (A) Properties of exonucleases tested in this study. +, strong activity; -, no significant activity; +/-, reduced activity; ss, single stranded; ds, double stranded; endo, endonuclease activity; dNMP, deoxyribonucleoside monophosphate; oligos, oligonucleotides. (B) Copies (3 × 10 8 ) of cell culture-derived viral DNA containing rcDNA and dslDNA were incubated for 1 h at 37°C with PSD (5 U), BAL-31 (5 U), Exo I (5 U), Exo V (5 U), and T5 Exo (5 U). Mung bean nuclease (5 U), EcoRI (5 U), and DNase I (5 U) were included as controls. After heat inactivation, the products were subjected to Southern blotting. The plasmid pUCX3.2 served as a marker for indicating the expected sizes of rcDNA and cccDNA.
    Figure Legend Snippet: Identification of exonucleases selectively digesting rcDNA. (A) Properties of exonucleases tested in this study. +, strong activity; -, no significant activity; +/-, reduced activity; ss, single stranded; ds, double stranded; endo, endonuclease activity; dNMP, deoxyribonucleoside monophosphate; oligos, oligonucleotides. (B) Copies (3 × 10 8 ) of cell culture-derived viral DNA containing rcDNA and dslDNA were incubated for 1 h at 37°C with PSD (5 U), BAL-31 (5 U), Exo I (5 U), Exo V (5 U), and T5 Exo (5 U). Mung bean nuclease (5 U), EcoRI (5 U), and DNase I (5 U) were included as controls. After heat inactivation, the products were subjected to Southern blotting. The plasmid pUCX3.2 served as a marker for indicating the expected sizes of rcDNA and cccDNA.

    Techniques Used: Activity Assay, Cell Culture, Derivative Assay, Incubation, Southern Blot, Plasmid Preparation, Marker

    30) Product Images from "T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR"

    Article Title: T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

    Journal: Journal of Virology

    doi: 10.1128/JVI.01117-18

    Identification of exonucleases selectively digesting rcDNA. (A) Properties of exonucleases tested in this study. +, strong activity; -, no significant activity; +/-, reduced activity; ss, single stranded; ds, double stranded; endo, endonuclease activity; dNMP, deoxyribonucleoside monophosphate; oligos, oligonucleotides. (B) Copies (3 × 10 8 ) of cell culture-derived viral DNA containing rcDNA and dslDNA were incubated for 1 h at 37°C with PSD (5 U), BAL-31 (5 U), Exo I (5 U), Exo V (5 U), and T5 Exo (5 U). Mung bean nuclease (5 U), EcoRI (5 U), and DNase I (5 U) were included as controls. After heat inactivation, the products were subjected to Southern blotting. The plasmid pUCX3.2 served as a marker for indicating the expected sizes of rcDNA and cccDNA.
    Figure Legend Snippet: Identification of exonucleases selectively digesting rcDNA. (A) Properties of exonucleases tested in this study. +, strong activity; -, no significant activity; +/-, reduced activity; ss, single stranded; ds, double stranded; endo, endonuclease activity; dNMP, deoxyribonucleoside monophosphate; oligos, oligonucleotides. (B) Copies (3 × 10 8 ) of cell culture-derived viral DNA containing rcDNA and dslDNA were incubated for 1 h at 37°C with PSD (5 U), BAL-31 (5 U), Exo I (5 U), Exo V (5 U), and T5 Exo (5 U). Mung bean nuclease (5 U), EcoRI (5 U), and DNase I (5 U) were included as controls. After heat inactivation, the products were subjected to Southern blotting. The plasmid pUCX3.2 served as a marker for indicating the expected sizes of rcDNA and cccDNA.

    Techniques Used: Activity Assay, Cell Culture, Derivative Assay, Incubation, Southern Blot, Plasmid Preparation, Marker

    Titration analysis of T5 Exo and PSD. (A) Two micrograms of genomic DNA samples from HBV-free HepG2 hNTCP cells was incubated with T5 Exo or PSD in time-dependent (1, 2, 4, and 16 h) and dose-dependent (1 and 5 U) manners. After digestion, products were visualized on an agarose gel, and the expression level of the human β-globin gene was measured as a representative readout to show digestion degree of genomic DNA. (B) Two micrograms of pSHH2.1 plasmid was incubated with T5 Exo or PSD similarly, and the remaining plasmid in products was determined by pp466-541 or directly visualized on an agarose gel.
    Figure Legend Snippet: Titration analysis of T5 Exo and PSD. (A) Two micrograms of genomic DNA samples from HBV-free HepG2 hNTCP cells was incubated with T5 Exo or PSD in time-dependent (1, 2, 4, and 16 h) and dose-dependent (1 and 5 U) manners. After digestion, products were visualized on an agarose gel, and the expression level of the human β-globin gene was measured as a representative readout to show digestion degree of genomic DNA. (B) Two micrograms of pSHH2.1 plasmid was incubated with T5 Exo or PSD similarly, and the remaining plasmid in products was determined by pp466-541 or directly visualized on an agarose gel.

    Techniques Used: Titration, Incubation, Agarose Gel Electrophoresis, Expressing, Plasmid Preparation

    T5 Exo efficiently removes rcDNA and genomic DNA from DNA preparation. (A) Copies (3 × 10 8 ) of virion DNA from purified HBV virions were incubated with PSD (5 U), T5 Exo (5 U), EcoRI (5 U), or DNase I (5 U) at 37°C for 1 h and further subjected to Southern blotting. pUCX3.2 plasmid (3.2 kb) was loaded as well to indicate the positions of rcDNA and cccDNA. (B) (Top) Two micrograms of purified 3.2-kb linear HBV monomer released from the pSHH2.1 plasmid by EcoRI digestion was incubated with indicated units of T5 Exo or PSD at 37°C for 1 h. (Middle) A mixture of 3.2-kb open circular DNA (2 μg) that was artificially nicked by Nb.BtsI endonuclease and 3.2-kb supercoiled pUCX3.2 plasmid (2 μg) was subjected to T5 Exo or PSD digestion at 37°C for 1 h. (Bottom) Two micrograms of genomic DNA from uninfected HepG2 hNTCP cells was similarly treated with T5 Exo or PSD. All digestion products are shown on agarose gels, and for relative quantification, band density of untreated samples is set as 100%. (C) Copies (10 8 ) of virion DNA or pUCX3.2 plasmid were digested with T5 Exo (5 U) or PSD (5 U) in the absence (0 μg) or presence (2 μg) of genomic DNA (as shown above; 1% agarose gel) at 37°C for 1 h, and the products were loaded for Southern blotting (bottom). (D) Virion DNA (rcV) or pSHH2.1 plasmid was incubated with T5 Exo (5 U) or PSD (10 U) at 37°C for 1 h, and products were further analyzed by pp466-541 (left) or pp1040-1996 (right), respectively. ns, no significance. (E) Total DNA samples from HBV-infected HepG2 hNTCP cells (days 1, 2, 3, 6, and 9 p.i. and day 0 without inocula) were incubated with T5 Exo (5 U) or PSD (10 U) as described above, and cccDNA (left) and total DNA (right) copies were quantified by respective primers.
    Figure Legend Snippet: T5 Exo efficiently removes rcDNA and genomic DNA from DNA preparation. (A) Copies (3 × 10 8 ) of virion DNA from purified HBV virions were incubated with PSD (5 U), T5 Exo (5 U), EcoRI (5 U), or DNase I (5 U) at 37°C for 1 h and further subjected to Southern blotting. pUCX3.2 plasmid (3.2 kb) was loaded as well to indicate the positions of rcDNA and cccDNA. (B) (Top) Two micrograms of purified 3.2-kb linear HBV monomer released from the pSHH2.1 plasmid by EcoRI digestion was incubated with indicated units of T5 Exo or PSD at 37°C for 1 h. (Middle) A mixture of 3.2-kb open circular DNA (2 μg) that was artificially nicked by Nb.BtsI endonuclease and 3.2-kb supercoiled pUCX3.2 plasmid (2 μg) was subjected to T5 Exo or PSD digestion at 37°C for 1 h. (Bottom) Two micrograms of genomic DNA from uninfected HepG2 hNTCP cells was similarly treated with T5 Exo or PSD. All digestion products are shown on agarose gels, and for relative quantification, band density of untreated samples is set as 100%. (C) Copies (10 8 ) of virion DNA or pUCX3.2 plasmid were digested with T5 Exo (5 U) or PSD (5 U) in the absence (0 μg) or presence (2 μg) of genomic DNA (as shown above; 1% agarose gel) at 37°C for 1 h, and the products were loaded for Southern blotting (bottom). (D) Virion DNA (rcV) or pSHH2.1 plasmid was incubated with T5 Exo (5 U) or PSD (10 U) at 37°C for 1 h, and products were further analyzed by pp466-541 (left) or pp1040-1996 (right), respectively. ns, no significance. (E) Total DNA samples from HBV-infected HepG2 hNTCP cells (days 1, 2, 3, 6, and 9 p.i. and day 0 without inocula) were incubated with T5 Exo (5 U) or PSD (10 U) as described above, and cccDNA (left) and total DNA (right) copies were quantified by respective primers.

    Techniques Used: Purification, Incubation, Southern Blot, Plasmid Preparation, Agarose Gel Electrophoresis, Infection

    Detection of cccDNA and validation of Myrcludex B in 96-well plate format. (A) HepG2 hNTCP cells seeded in a 96-well plate were infected at an mge/cell of 1,000. Myrcludex B was coadministered, tenofovir was added postinoculation, and IFN-α-2a was applied during and after infection. (B) Cells were treated with each antiviral at eight doses (1:3.2 serial dilutions) in triplicates. (C) On day 7 p.i., DNA samples were extracted together using a vacuum-based system. Crude DNA in 100 μl of elute was ethanol precipitated and resuspended with 10 μl of water. T5 Exo digestion was performed prior to cccDNA quantification by PCR. HBeAg levels during days 5 to 7 p.i. in the supernatant in all wells were measured.
    Figure Legend Snippet: Detection of cccDNA and validation of Myrcludex B in 96-well plate format. (A) HepG2 hNTCP cells seeded in a 96-well plate were infected at an mge/cell of 1,000. Myrcludex B was coadministered, tenofovir was added postinoculation, and IFN-α-2a was applied during and after infection. (B) Cells were treated with each antiviral at eight doses (1:3.2 serial dilutions) in triplicates. (C) On day 7 p.i., DNA samples were extracted together using a vacuum-based system. Crude DNA in 100 μl of elute was ethanol precipitated and resuspended with 10 μl of water. T5 Exo digestion was performed prior to cccDNA quantification by PCR. HBeAg levels during days 5 to 7 p.i. in the supernatant in all wells were measured.

    Techniques Used: Infection, Polymerase Chain Reaction

    cccDNA profiles in infections with increasing mge. (A) HepG2 hNTCP cells were infected with different amounts of virus inoculum (mge/cell of 30, 100, 300, 1,000, and 3,000) in parallel, and total DNA samples were prepared on day 10 p.i. Samples were hydrolyzed by T5 Exo (5 U, 60 min) at 37°C for 1 h, and cccDNA was determined using pp1040-1996. Total DNA copy numbers were also determined in undigested samples using pp466-541. (B) Within the same infections, secreted HBsAg values from day 7 to 10 p.i. were detected. (C) On day 10 p.i., intracellular HBcAg expression levels (red) were visualized. As a control to verify NTCP-mediated entry of the virus, Myrcludex B (1 μM) was administered during the infection.
    Figure Legend Snippet: cccDNA profiles in infections with increasing mge. (A) HepG2 hNTCP cells were infected with different amounts of virus inoculum (mge/cell of 30, 100, 300, 1,000, and 3,000) in parallel, and total DNA samples were prepared on day 10 p.i. Samples were hydrolyzed by T5 Exo (5 U, 60 min) at 37°C for 1 h, and cccDNA was determined using pp1040-1996. Total DNA copy numbers were also determined in undigested samples using pp466-541. (B) Within the same infections, secreted HBsAg values from day 7 to 10 p.i. were detected. (C) On day 10 p.i., intracellular HBcAg expression levels (red) were visualized. As a control to verify NTCP-mediated entry of the virus, Myrcludex B (1 μM) was administered during the infection.

    Techniques Used: Infection, Expressing

    T5 Exo and Exo III remove HBV replicative intermediates without affecting cccDNA. HepG2 hNTCP cells were seeded in a 6-well plate and infected at an mge/cell of 3,000. To block entry, Myrcludex B (2 μM) was used as a control. (A) On day 7 p.i., cytosolic DNA samples were extracted as described in Materials and Methods and hydrolyzed by Exo I (5 U, 60 min), Exo III (25 U, 60 min), Exo I and III (5 U plus 25 U, 60 min), T5 Exo (5 U, 60 min), PSD (10 U, 60 min), and EcoRI (10 U, 60 min) at 37°C for 1 h, and later on, all enzymes were heat denatured at 70°C. Samples were analyzed by Southern blotting (left) and PCR with pp466-541 (right). (B) HepG2 hNTCP cells were infected in a 6-well plate format for 7 days, and the DNA samples were Hirt extracted and hydrolyzed by the respective enzymes prior to Southern blotting (left) and cccDNA-specific PCR using pp1040-1996 (right).
    Figure Legend Snippet: T5 Exo and Exo III remove HBV replicative intermediates without affecting cccDNA. HepG2 hNTCP cells were seeded in a 6-well plate and infected at an mge/cell of 3,000. To block entry, Myrcludex B (2 μM) was used as a control. (A) On day 7 p.i., cytosolic DNA samples were extracted as described in Materials and Methods and hydrolyzed by Exo I (5 U, 60 min), Exo III (25 U, 60 min), Exo I and III (5 U plus 25 U, 60 min), T5 Exo (5 U, 60 min), PSD (10 U, 60 min), and EcoRI (10 U, 60 min) at 37°C for 1 h, and later on, all enzymes were heat denatured at 70°C. Samples were analyzed by Southern blotting (left) and PCR with pp466-541 (right). (B) HepG2 hNTCP cells were infected in a 6-well plate format for 7 days, and the DNA samples were Hirt extracted and hydrolyzed by the respective enzymes prior to Southern blotting (left) and cccDNA-specific PCR using pp1040-1996 (right).

    Techniques Used: Infection, Blocking Assay, Southern Blot, Polymerase Chain Reaction

    31) Product Images from "T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR"

    Article Title: T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

    Journal: Journal of Virology

    doi: 10.1128/JVI.01117-18

    T5 Exo and Exo III remove HBV replicative intermediates without affecting cccDNA. HepG2 hNTCP cells were seeded in a 6-well plate and infected at an mge/cell of 3,000. To block entry, Myrcludex B (2 μM) was used as a control. (A) On day 7 p.i., cytosolic DNA samples were extracted as described in Materials and Methods and hydrolyzed by Exo I (5 U, 60 min), Exo III (25 U, 60 min), Exo I and III (5 U plus 25 U, 60 min), T5 Exo (5 U, 60 min), PSD (10 U, 60 min), and EcoRI (10 U, 60 min) at 37°C for 1 h, and later on, all enzymes were heat denatured at 70°C. Samples were analyzed by Southern blotting (left) and PCR with pp466-541 (right). (B) HepG2 hNTCP cells were infected in a 6-well plate format for 7 days, and the DNA samples were Hirt extracted and hydrolyzed by the respective enzymes prior to Southern blotting (left) and cccDNA-specific PCR using pp1040-1996 (right).
    Figure Legend Snippet: T5 Exo and Exo III remove HBV replicative intermediates without affecting cccDNA. HepG2 hNTCP cells were seeded in a 6-well plate and infected at an mge/cell of 3,000. To block entry, Myrcludex B (2 μM) was used as a control. (A) On day 7 p.i., cytosolic DNA samples were extracted as described in Materials and Methods and hydrolyzed by Exo I (5 U, 60 min), Exo III (25 U, 60 min), Exo I and III (5 U plus 25 U, 60 min), T5 Exo (5 U, 60 min), PSD (10 U, 60 min), and EcoRI (10 U, 60 min) at 37°C for 1 h, and later on, all enzymes were heat denatured at 70°C. Samples were analyzed by Southern blotting (left) and PCR with pp466-541 (right). (B) HepG2 hNTCP cells were infected in a 6-well plate format for 7 days, and the DNA samples were Hirt extracted and hydrolyzed by the respective enzymes prior to Southern blotting (left) and cccDNA-specific PCR using pp1040-1996 (right).

    Techniques Used: Infection, Blocking Assay, Southern Blot, Polymerase Chain Reaction

    32) Product Images from "Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles"

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp137

    The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.
    Figure Legend Snippet: The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Ligation, Migration

    Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).
    Figure Legend Snippet: Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).

    Techniques Used: Agarose Gel Electrophoresis

    33) Product Images from "Design and characterization of decoy oligonucleotides containing locked nucleic acids"

    Article Title: Design and characterization of decoy oligonucleotides containing locked nucleic acids

    Journal: Nucleic Acids Research

    doi:

    Susceptibility to BAL-31 nuclease degradation of LNA-modified ODNs. LNA-modified [NF-κB(a), (b), (c), (c+b) and (b+c)] and control phosphodiester [NF-κB(d)] decoy molecules were incubated for different lengths of time, as indicated, with 0.5 U/ml BAL-31 and then submitted to electrophoretic separation on 2.5% (w/v) agarose gels. Detection and quantitation of the ethidium bromide stained bands were performed in a Molecular Analyst. Volume densities of the bands are expressed as percent ODN decoy remaining with respect to the relative time zero value and are shown as line graphs.
    Figure Legend Snippet: Susceptibility to BAL-31 nuclease degradation of LNA-modified ODNs. LNA-modified [NF-κB(a), (b), (c), (c+b) and (b+c)] and control phosphodiester [NF-κB(d)] decoy molecules were incubated for different lengths of time, as indicated, with 0.5 U/ml BAL-31 and then submitted to electrophoretic separation on 2.5% (w/v) agarose gels. Detection and quantitation of the ethidium bromide stained bands were performed in a Molecular Analyst. Volume densities of the bands are expressed as percent ODN decoy remaining with respect to the relative time zero value and are shown as line graphs.

    Techniques Used: Modification, Incubation, Quantitation Assay, Staining

    34) Product Images from "Design and characterization of decoy oligonucleotides containing locked nucleic acids"

    Article Title: Design and characterization of decoy oligonucleotides containing locked nucleic acids

    Journal: Nucleic Acids Research

    doi:

    Susceptibility to BAL-31 nuclease degradation of LNA-modified ODNs. LNA-modified [NF-κB(a), (b), (c), (c+b) and (b+c)] and control phosphodiester [NF-κB(d)] decoy molecules were incubated for different lengths of time, as indicated, with 0.5 U/ml BAL-31 and then submitted to electrophoretic separation on 2.5% (w/v) agarose gels. Detection and quantitation of the ethidium bromide stained bands were performed in a Molecular Analyst. Volume densities of the bands are expressed as percent ODN decoy remaining with respect to the relative time zero value and are shown as line graphs.
    Figure Legend Snippet: Susceptibility to BAL-31 nuclease degradation of LNA-modified ODNs. LNA-modified [NF-κB(a), (b), (c), (c+b) and (b+c)] and control phosphodiester [NF-κB(d)] decoy molecules were incubated for different lengths of time, as indicated, with 0.5 U/ml BAL-31 and then submitted to electrophoretic separation on 2.5% (w/v) agarose gels. Detection and quantitation of the ethidium bromide stained bands were performed in a Molecular Analyst. Volume densities of the bands are expressed as percent ODN decoy remaining with respect to the relative time zero value and are shown as line graphs.

    Techniques Used: Modification, Incubation, Quantitation Assay, Staining

    35) Product Images from "Novel Virulent and Broad-Host-Range Erwinia amylovora Bacteriophages Reveal a High Degree of Mosaicism and a Relationship to Enterobacteriaceae Phages ▿ Phages ▿ †"

    Article Title: Novel Virulent and Broad-Host-Range Erwinia amylovora Bacteriophages Reveal a High Degree of Mosaicism and a Relationship to Enterobacteriaceae Phages ▿ Phages ▿ †

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.03022-10

    Analysis of phage genome physical structures. Shown are fragment patterns of phage DNA, following time-limited BAL31 treatment and subsequent digestion with different endonucleases. Phage L1 was digested with MfeI, M7 with FspBI, S6 with three different
    Figure Legend Snippet: Analysis of phage genome physical structures. Shown are fragment patterns of phage DNA, following time-limited BAL31 treatment and subsequent digestion with different endonucleases. Phage L1 was digested with MfeI, M7 with FspBI, S6 with three different

    Techniques Used:

    36) Product Images from "Cooperative kinking at distant sites in mechanically stressed DNA"

    Article Title: Cooperative kinking at distant sites in mechanically stressed DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr666

    DNA minicircles with 100, 106 and 108 bp, their purity and their susceptibility to Bal31 digestion. In a 10% denaturing PAGE gel (7 M urea), the denatured linear DNA fragments used as a marker, migrate much quicker than circular, covalently closed DNA minicircles in which the two strands cannot separate in space. Notice the presence of only one topoisomer in each category of minicircles and the sensitivity of minicircles with 100 bp to Bal31 nuclease.
    Figure Legend Snippet: DNA minicircles with 100, 106 and 108 bp, their purity and their susceptibility to Bal31 digestion. In a 10% denaturing PAGE gel (7 M urea), the denatured linear DNA fragments used as a marker, migrate much quicker than circular, covalently closed DNA minicircles in which the two strands cannot separate in space. Notice the presence of only one topoisomer in each category of minicircles and the sensitivity of minicircles with 100 bp to Bal31 nuclease.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Marker

    37) Product Images from "A shotgun antisense approach to the identification of novel essential genes in Pseudomonas aeruginosa"

    Article Title: A shotgun antisense approach to the identification of novel essential genes in Pseudomonas aeruginosa

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-14-24

    Construction and screening of PAO1 SALs. (A) Genomic DNA was isolated from P. aeruginosa PAO1 and nebulized to obtain sheared fragments of 200–800 bp. After treatment with exonuclease BAL-31 and Klenow polymerase, the genomic DNA fragments were cloned into the E. coli strain JM109, downstream of the arabinose-inducible promoter P BAD of the pHERD20T vector. (B) E. coli transformants, representing the PAO1 shotgun antisense library (SAL), were arrayed in 96-well microplates and (C) mated with P. aeruginosa PAO1 in the presence of a helper strain (triparental mating). (D) SAL recipient PAO1 exconjugants were selected by spotting on PIA plates supplemented with Cb both in the absence and in the presence of the P BAD inducer arabinose. Recipient PAO1 exconjugant spots were inspected for growth defects following 24 h of incubation at 37°C. (E) The identity of the genomic fragments eliciting growth defects (lethal effects, indicated by a lack of a spot: only with inducer, e.g. clones A4, A8, B5, and E4, and with and without an inducer, e.g. clones A2 and E6; growth impairment, indicated as gray spots: only with an inducer, e.g. clones C2, A6, and B6, and with and without an inducer, e.g. C3 and B8) was determined by sequencing the inserts in the corresponding clones of E. coli SAL.
    Figure Legend Snippet: Construction and screening of PAO1 SALs. (A) Genomic DNA was isolated from P. aeruginosa PAO1 and nebulized to obtain sheared fragments of 200–800 bp. After treatment with exonuclease BAL-31 and Klenow polymerase, the genomic DNA fragments were cloned into the E. coli strain JM109, downstream of the arabinose-inducible promoter P BAD of the pHERD20T vector. (B) E. coli transformants, representing the PAO1 shotgun antisense library (SAL), were arrayed in 96-well microplates and (C) mated with P. aeruginosa PAO1 in the presence of a helper strain (triparental mating). (D) SAL recipient PAO1 exconjugants were selected by spotting on PIA plates supplemented with Cb both in the absence and in the presence of the P BAD inducer arabinose. Recipient PAO1 exconjugant spots were inspected for growth defects following 24 h of incubation at 37°C. (E) The identity of the genomic fragments eliciting growth defects (lethal effects, indicated by a lack of a spot: only with inducer, e.g. clones A4, A8, B5, and E4, and with and without an inducer, e.g. clones A2 and E6; growth impairment, indicated as gray spots: only with an inducer, e.g. clones C2, A6, and B6, and with and without an inducer, e.g. C3 and B8) was determined by sequencing the inserts in the corresponding clones of E. coli SAL.

    Techniques Used: Isolation, Clone Assay, Plasmid Preparation, Incubation, Sequencing

    38) Product Images from "T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR"

    Article Title: T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

    Journal: Journal of Virology

    doi: 10.1128/JVI.01117-18

    T5 Exo efficiently removes rcDNA and genomic DNA from DNA preparation. (A) Copies (3 × 10 8 ) of virion DNA from purified HBV virions were incubated with PSD (5 U), T5 Exo (5 U), EcoRI (5 U), or DNase I (5 U) at 37°C for 1 h and further subjected to Southern blotting. pUCX3.2 plasmid (3.2 kb) was loaded as well to indicate the positions of rcDNA and cccDNA. (B) (Top) Two micrograms of purified 3.2-kb linear HBV monomer released from the pSHH2.1 plasmid by EcoRI digestion was incubated with indicated units of T5 Exo or PSD at 37°C for 1 h. (Middle) A mixture of 3.2-kb open circular DNA (2 μg) that was artificially nicked by Nb.BtsI endonuclease and 3.2-kb supercoiled pUCX3.2 plasmid (2 μg) was subjected to T5 Exo or PSD digestion at 37°C for 1 h. (Bottom) Two micrograms of genomic DNA from uninfected HepG2 hNTCP cells was similarly treated with T5 Exo or PSD. All digestion products are shown on agarose gels, and for relative quantification, band density of untreated samples is set as 100%. (C) Copies (10 8 ) of virion DNA or pUCX3.2 plasmid were digested with T5 Exo (5 U) or PSD (5 U) in the absence (0 μg) or presence (2 μg) of genomic DNA (as shown above; 1% agarose gel) at 37°C for 1 h, and the products were loaded for Southern blotting (bottom). (D) Virion DNA (rcV) or pSHH2.1 plasmid was incubated with T5 Exo (5 U) or PSD (10 U) at 37°C for 1 h, and products were further analyzed by pp466-541 (left) or pp1040-1996 (right), respectively. ns, no significance. (E) Total DNA samples from HBV-infected HepG2 hNTCP cells (days 1, 2, 3, 6, and 9 p.i. and day 0 without inocula) were incubated with T5 Exo (5 U) or PSD (10 U) as described above, and cccDNA (left) and total DNA (right) copies were quantified by respective primers.
    Figure Legend Snippet: T5 Exo efficiently removes rcDNA and genomic DNA from DNA preparation. (A) Copies (3 × 10 8 ) of virion DNA from purified HBV virions were incubated with PSD (5 U), T5 Exo (5 U), EcoRI (5 U), or DNase I (5 U) at 37°C for 1 h and further subjected to Southern blotting. pUCX3.2 plasmid (3.2 kb) was loaded as well to indicate the positions of rcDNA and cccDNA. (B) (Top) Two micrograms of purified 3.2-kb linear HBV monomer released from the pSHH2.1 plasmid by EcoRI digestion was incubated with indicated units of T5 Exo or PSD at 37°C for 1 h. (Middle) A mixture of 3.2-kb open circular DNA (2 μg) that was artificially nicked by Nb.BtsI endonuclease and 3.2-kb supercoiled pUCX3.2 plasmid (2 μg) was subjected to T5 Exo or PSD digestion at 37°C for 1 h. (Bottom) Two micrograms of genomic DNA from uninfected HepG2 hNTCP cells was similarly treated with T5 Exo or PSD. All digestion products are shown on agarose gels, and for relative quantification, band density of untreated samples is set as 100%. (C) Copies (10 8 ) of virion DNA or pUCX3.2 plasmid were digested with T5 Exo (5 U) or PSD (5 U) in the absence (0 μg) or presence (2 μg) of genomic DNA (as shown above; 1% agarose gel) at 37°C for 1 h, and the products were loaded for Southern blotting (bottom). (D) Virion DNA (rcV) or pSHH2.1 plasmid was incubated with T5 Exo (5 U) or PSD (10 U) at 37°C for 1 h, and products were further analyzed by pp466-541 (left) or pp1040-1996 (right), respectively. ns, no significance. (E) Total DNA samples from HBV-infected HepG2 hNTCP cells (days 1, 2, 3, 6, and 9 p.i. and day 0 without inocula) were incubated with T5 Exo (5 U) or PSD (10 U) as described above, and cccDNA (left) and total DNA (right) copies were quantified by respective primers.

    Techniques Used: Purification, Incubation, Southern Blot, Plasmid Preparation, Agarose Gel Electrophoresis, Infection

    T5 Exo and Exo III remove HBV replicative intermediates without affecting cccDNA. HepG2 hNTCP cells were seeded in a 6-well plate and infected at an mge/cell of 3,000. To block entry, Myrcludex B (2 μM) was used as a control. (A) On day 7 p.i., cytosolic DNA samples were extracted as described in Materials and Methods and hydrolyzed by Exo I (5 U, 60 min), Exo III (25 U, 60 min), Exo I and III (5 U plus 25 U, 60 min), T5 Exo (5 U, 60 min), PSD (10 U, 60 min), and EcoRI (10 U, 60 min) at 37°C for 1 h, and later on, all enzymes were heat denatured at 70°C. Samples were analyzed by Southern blotting (left) and PCR with pp466-541 (right). (B) HepG2 hNTCP cells were infected in a 6-well plate format for 7 days, and the DNA samples were Hirt extracted and hydrolyzed by the respective enzymes prior to Southern blotting (left) and cccDNA-specific PCR using pp1040-1996 (right).
    Figure Legend Snippet: T5 Exo and Exo III remove HBV replicative intermediates without affecting cccDNA. HepG2 hNTCP cells were seeded in a 6-well plate and infected at an mge/cell of 3,000. To block entry, Myrcludex B (2 μM) was used as a control. (A) On day 7 p.i., cytosolic DNA samples were extracted as described in Materials and Methods and hydrolyzed by Exo I (5 U, 60 min), Exo III (25 U, 60 min), Exo I and III (5 U plus 25 U, 60 min), T5 Exo (5 U, 60 min), PSD (10 U, 60 min), and EcoRI (10 U, 60 min) at 37°C for 1 h, and later on, all enzymes were heat denatured at 70°C. Samples were analyzed by Southern blotting (left) and PCR with pp466-541 (right). (B) HepG2 hNTCP cells were infected in a 6-well plate format for 7 days, and the DNA samples were Hirt extracted and hydrolyzed by the respective enzymes prior to Southern blotting (left) and cccDNA-specific PCR using pp1040-1996 (right).

    Techniques Used: Infection, Blocking Assay, Southern Blot, Polymerase Chain Reaction

    39) Product Images from "Selaginella moellendorffii telomeres: conserved and unique features in an ancient land plant lineage"

    Article Title: Selaginella moellendorffii telomeres: conserved and unique features in an ancient land plant lineage

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2012.00161

    Telomere length analysis in Selaginella moellendorffii . (A) Comparative terminal restriction fragment (TRF) analysis of S. moellendorffii (lane 1) and A. thaliana (lane 2) telomeres. Molecular weight markers are shown on the left. (B) Bal 31 digestion of S. moellendorffii telomeric DNA. Lane 1, Tru 1I digestion of genomic DNA without prior Bal treatment (0 min). Lanes 2–6, Tru 1I digestion of genomic DNA with Bal 31 tr31eatment for 15, 30, 45, 60, and 90 min, respectively. Asterisks indicate cross-hybridizing interstitial telomeric DNA bands, which are not sensitive to Bal 31 digestion for up to 90 min.
    Figure Legend Snippet: Telomere length analysis in Selaginella moellendorffii . (A) Comparative terminal restriction fragment (TRF) analysis of S. moellendorffii (lane 1) and A. thaliana (lane 2) telomeres. Molecular weight markers are shown on the left. (B) Bal 31 digestion of S. moellendorffii telomeric DNA. Lane 1, Tru 1I digestion of genomic DNA without prior Bal treatment (0 min). Lanes 2–6, Tru 1I digestion of genomic DNA with Bal 31 tr31eatment for 15, 30, 45, 60, and 90 min, respectively. Asterisks indicate cross-hybridizing interstitial telomeric DNA bands, which are not sensitive to Bal 31 digestion for up to 90 min.

    Techniques Used: Molecular Weight

    40) Product Images from "Cooperative kinking at distant sites in mechanically stressed DNA"

    Article Title: Cooperative kinking at distant sites in mechanically stressed DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr666

    DNA minicircles with 100, 106 and 108 bp, their purity and their susceptibility to Bal31 digestion. In a 10% denaturing PAGE gel (7 M urea), the denatured linear DNA fragments used as a marker, migrate much quicker than circular, covalently closed DNA minicircles in which the two strands cannot separate in space. Notice the presence of only one topoisomer in each category of minicircles and the sensitivity of minicircles with 100 bp to Bal31 nuclease.
    Figure Legend Snippet: DNA minicircles with 100, 106 and 108 bp, their purity and their susceptibility to Bal31 digestion. In a 10% denaturing PAGE gel (7 M urea), the denatured linear DNA fragments used as a marker, migrate much quicker than circular, covalently closed DNA minicircles in which the two strands cannot separate in space. Notice the presence of only one topoisomer in each category of minicircles and the sensitivity of minicircles with 100 bp to Bal31 nuclease.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Marker

    Related Articles

    Sequencing:

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles
    Article Snippet: .. However, the 94-bp-long minicircles with the sequence selected for their very efficient cyclization by C & W were not previously tested for their sensitivity to Bal31 nuclease. ..

    Incubation:

    Article Title: Telomere length is highly heritable and independent of growth rate manipulated by temperature in field crickets
    Article Snippet: .. The other 3 pieces were incubated for 20 minutes (T=20), 80 minutes (T=80) and 240 minutes (T=240) respectively, with 200μl 0.1U Nuclease Bal 31 enzyme (New England Biolabs #M0213S) at 30°C, immediately followed by washing similar to T=0 to stop the digestion. ..

    other:

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles
    Article Snippet: Du et al . showed earlier that 63-bp DNA minicircles are so strongly bent that they form kinks and become sensitive to Bal31 nuclease, while 85-bp DNA minicircles can accommodate their bending stress without kinks and without becoming sensitive to Bal31 nuclease ( ).

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles
    Article Snippet: However, torsionally relaxed extremely small DNA minicircles having only 63 base pairs were digested by Bal31 nuclease.

    Article Title: Production of DNA minicircles less than 250 base pairs through a novel concentrated DNA circularization assay enabling minicircle design with NF-κB inhibition activity
    Article Snippet: We next used Bal31 nuclease to detect the presence of DNA distortions within our constrained minicircles.

    Article Title: Cooperative kinking at distant sites in mechanically stressed DNA
    Article Snippet: This result indicates that the negative torsional stress sustained within MC100, previously shown to result in sensitivity to Bal31 nuclease, also leads to the creation of easily bendable sites that permit the molecules to minimize their bending energy by adopting elongated, elliptical shapes.

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    New England Biolabs bal31 nuclease
    DNA minicircles with 100, 106 and 108 bp, their purity and their susceptibility to <t>Bal31</t> digestion. In a 10% denaturing PAGE gel (7 M urea), the denatured linear DNA fragments used as a marker, migrate much quicker than circular, covalently closed DNA minicircles in which the two strands cannot separate in space. Notice the presence of only one topoisomer in each category of minicircles and the sensitivity of minicircles with 100 bp to Bal31 nuclease.
    Bal31 Nuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DNA minicircles with 100, 106 and 108 bp, their purity and their susceptibility to Bal31 digestion. In a 10% denaturing PAGE gel (7 M urea), the denatured linear DNA fragments used as a marker, migrate much quicker than circular, covalently closed DNA minicircles in which the two strands cannot separate in space. Notice the presence of only one topoisomer in each category of minicircles and the sensitivity of minicircles with 100 bp to Bal31 nuclease.

    Journal: Nucleic Acids Research

    Article Title: Cooperative kinking at distant sites in mechanically stressed DNA

    doi: 10.1093/nar/gkr666

    Figure Lengend Snippet: DNA minicircles with 100, 106 and 108 bp, their purity and their susceptibility to Bal31 digestion. In a 10% denaturing PAGE gel (7 M urea), the denatured linear DNA fragments used as a marker, migrate much quicker than circular, covalently closed DNA minicircles in which the two strands cannot separate in space. Notice the presence of only one topoisomer in each category of minicircles and the sensitivity of minicircles with 100 bp to Bal31 nuclease.

    Article Snippet: This result indicates that the negative torsional stress sustained within MC100, previously shown to result in sensitivity to Bal31 nuclease, also leads to the creation of easily bendable sites that permit the molecules to minimize their bending energy by adopting elongated, elliptical shapes.

    Techniques: Polyacrylamide Gel Electrophoresis, Marker

    The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.

    Journal: Nucleic Acids Research

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles

    doi: 10.1093/nar/gkp137

    Figure Lengend Snippet: The formation and purification of 94-bp-long covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that after the ligation reaction of minicircles with nicks, new species appear on the gel (compare lanes 3 with 2). Only these new species resist digestion by Bal31 (lane 4) indicating that they are covalently closed DNA molecules. The electrophoretic migration of these new species is as expected for monomeric, dimeric and trimeric rings that acquired writhe due to ethidium bromide intercalation to covalently closed DNA molecules.

    Article Snippet: However, the 94-bp-long minicircles with the sequence selected for their very efficient cyclization by C & W were not previously tested for their sensitivity to Bal31 nuclease.

    Techniques: Purification, Agarose Gel Electrophoresis, Ligation, Migration

    Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).

    Journal: Nucleic Acids Research

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles

    doi: 10.1093/nar/gkp137

    Figure Lengend Snippet: Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).

    Article Snippet: However, the 94-bp-long minicircles with the sequence selected for their very efficient cyclization by C & W were not previously tested for their sensitivity to Bal31 nuclease.

    Techniques: Agarose Gel Electrophoresis

    CNDCA-based production of supercoiled minicircle. Image of stained native polyacrylamide gel showing 95 bp minicircle topoisomers of decreased linking number (ΔLk values of −1 and −2) (lanes 4, 6) as indicated on the right and obtained by religation of the nicked minicircle in the presence of EtBr. The slowest migrating band corresponds to the relaxed minicircle that migrates expectedly at the same rate as the nicked minicircle (lanes 1 and 2). Digestion of minicircle samples by the nuclease Bal31 is indicated on the right. The position of the molecular mass markers in bp is indicated on the left.

    Journal: Nucleic Acids Research

    Article Title: Production of DNA minicircles less than 250 base pairs through a novel concentrated DNA circularization assay enabling minicircle design with NF-κB inhibition activity

    doi: 10.1093/nar/gkw1034

    Figure Lengend Snippet: CNDCA-based production of supercoiled minicircle. Image of stained native polyacrylamide gel showing 95 bp minicircle topoisomers of decreased linking number (ΔLk values of −1 and −2) (lanes 4, 6) as indicated on the right and obtained by religation of the nicked minicircle in the presence of EtBr. The slowest migrating band corresponds to the relaxed minicircle that migrates expectedly at the same rate as the nicked minicircle (lanes 1 and 2). Digestion of minicircle samples by the nuclease Bal31 is indicated on the right. The position of the molecular mass markers in bp is indicated on the left.

    Article Snippet: We next used Bal31 nuclease to detect the presence of DNA distortions within our constrained minicircles.

    Techniques: Staining

    Pulsed field gel electrophoresis showing TTAGG n telomere restriction fragments from the denatured chromosomes of six individual crickets (indicated by the numbers 1-6). XV molecular size ladders are shown on the outer lanes and shorter 1kb ladders are shown between crickets 2-3 and 4-5. The photo shows four aliquots of DNA sample per cricket and these aliquots were exposed to 0 (i.e. untreated), 20, 80, and 240 minutes of Bal 31 . Each time series clearly shows that Bal 31 decreases the molecular size distribution of TTAGG fragments. The absence of a banded distribution in the smears suggest that interstitial telomeric repeats were not present and that chromosome strands were intact. Taken together, these findings show that TTAGG n fragments were located at the chromosome-ends.

    Journal: bioRxiv

    Article Title: Telomere length is highly heritable and independent of growth rate manipulated by temperature in field crickets

    doi: 10.1101/2020.05.29.123216

    Figure Lengend Snippet: Pulsed field gel electrophoresis showing TTAGG n telomere restriction fragments from the denatured chromosomes of six individual crickets (indicated by the numbers 1-6). XV molecular size ladders are shown on the outer lanes and shorter 1kb ladders are shown between crickets 2-3 and 4-5. The photo shows four aliquots of DNA sample per cricket and these aliquots were exposed to 0 (i.e. untreated), 20, 80, and 240 minutes of Bal 31 . Each time series clearly shows that Bal 31 decreases the molecular size distribution of TTAGG fragments. The absence of a banded distribution in the smears suggest that interstitial telomeric repeats were not present and that chromosome strands were intact. Taken together, these findings show that TTAGG n fragments were located at the chromosome-ends.

    Article Snippet: The other 3 pieces were incubated for 20 minutes (T=20), 80 minutes (T=80) and 240 minutes (T=240) respectively, with 200μl 0.1U Nuclease Bal 31 enzyme (New England Biolabs #M0213S) at 30°C, immediately followed by washing similar to T=0 to stop the digestion.

    Techniques: Pulsed-Field Gel, Electrophoresis