hindiii restriction endonucleases  (New England Biolabs)


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    Name:
    HindIII
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    HindIII 50 000 units
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    r0104l
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    50 000 units
    Category:
    Restriction Enzymes
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    New England Biolabs hindiii restriction endonucleases
    HindIII
    HindIII 50 000 units
    https://www.bioz.com/result/hindiii restriction endonucleases/product/New England Biolabs
    Average 99 stars, based on 996 article reviews
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    hindiii restriction endonucleases - by Bioz Stars, 2021-02
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    Images

    1) Product Images from "Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases"

    Article Title: Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases

    Journal: eLife

    doi: 10.7554/eLife.18574

    hDNA2 D277A unwinds plasmid- and oligonucleotide-based DNA substrates. ( A ) Representative 1% agarose gel showing hDNA2 D277A helicase activity on a λDNA/HindIII substrate in a time-course experiment with 346 nM hRPA. Heat, heat-denatured DNA substrate. ( B ) Representative 1% agarose gel showing that nuclease- and helicase-deficient hDNA2 D277A K654R (lanes 2–6) and helicase-deficient hDNA2 K654R (lane 8) do not exhibit helicase activity. Lane 7, DNA unwinding by nuclease-deficient DNA2 D277A. Reactions contained 215 nM hRPA. ( C – E ) Representative 10% polyacrylamide gels showing the helicase activity of hDNA2 D277A with ( C ) 5’ overhang, ( D ) 3’ overhang and with ( E ) dsDNA substrates. Reactions contained 7.5 nM RPA. Heat, heat-denatured DNA substrate. ( F ) Representative 1% agarose gels showing DNA unwinding of a 2.7 kbp-long substrate by either hDNA2 D277A (left part, at 37°C) or yDna2 E675A (right part, at 30°C) in a kinetic experiment with 215 nM human RPA or 267 nM yeast RPA respectively. ( G ) Quantitation of experiments such as shown in F. Averages shown, n = 2; error bars, SEM. DOI: http://dx.doi.org/10.7554/eLife.18574.007
    Figure Legend Snippet: hDNA2 D277A unwinds plasmid- and oligonucleotide-based DNA substrates. ( A ) Representative 1% agarose gel showing hDNA2 D277A helicase activity on a λDNA/HindIII substrate in a time-course experiment with 346 nM hRPA. Heat, heat-denatured DNA substrate. ( B ) Representative 1% agarose gel showing that nuclease- and helicase-deficient hDNA2 D277A K654R (lanes 2–6) and helicase-deficient hDNA2 K654R (lane 8) do not exhibit helicase activity. Lane 7, DNA unwinding by nuclease-deficient DNA2 D277A. Reactions contained 215 nM hRPA. ( C – E ) Representative 10% polyacrylamide gels showing the helicase activity of hDNA2 D277A with ( C ) 5’ overhang, ( D ) 3’ overhang and with ( E ) dsDNA substrates. Reactions contained 7.5 nM RPA. Heat, heat-denatured DNA substrate. ( F ) Representative 1% agarose gels showing DNA unwinding of a 2.7 kbp-long substrate by either hDNA2 D277A (left part, at 37°C) or yDna2 E675A (right part, at 30°C) in a kinetic experiment with 215 nM human RPA or 267 nM yeast RPA respectively. ( G ) Quantitation of experiments such as shown in F. Averages shown, n = 2; error bars, SEM. DOI: http://dx.doi.org/10.7554/eLife.18574.007

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Activity Assay, Recombinase Polymerase Amplification, Quantitation Assay

    2) Product Images from "Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma"

    Article Title: Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-020-01400-6

    a Agarose gel-electrophoresis of nucleic acids isolated from purified hIFNγ IBs. 1 —Sample, isolated from IBs, b Agarose gel-electrophoresis of nucleic acids isolated from purified hIFNγ IBs treated with restriction endonuclease XhoI and c HindIII. 1 —Non-treated sample; 2(b) —Sample treated with XhoI; 2(c) —sample treated with HindIII; d Enzymatic digestion of nucleic acids isolated from purified hIFNγ IBs 1 —non treated sample; 2 —sample treated with mixture of RNase A and RNase T 1 ; 3 —sample treated with DNase I; 4 —sample treated with Proteinase K; M —Molecular weight marker, bp
    Figure Legend Snippet: a Agarose gel-electrophoresis of nucleic acids isolated from purified hIFNγ IBs. 1 —Sample, isolated from IBs, b Agarose gel-electrophoresis of nucleic acids isolated from purified hIFNγ IBs treated with restriction endonuclease XhoI and c HindIII. 1 —Non-treated sample; 2(b) —Sample treated with XhoI; 2(c) —sample treated with HindIII; d Enzymatic digestion of nucleic acids isolated from purified hIFNγ IBs 1 —non treated sample; 2 —sample treated with mixture of RNase A and RNase T 1 ; 3 —sample treated with DNase I; 4 —sample treated with Proteinase K; M —Molecular weight marker, bp

    Techniques Used: Agarose Gel Electrophoresis, Isolation, Purification, Molecular Weight, Marker

    3) Product Images from "Liver fluke infections by Amphimerus sp. (Digenea: Opisthorchiidae) in definitive and fish intermediate hosts in Manabí province, Ecuador"

    Article Title: Liver fluke infections by Amphimerus sp. (Digenea: Opisthorchiidae) in definitive and fish intermediate hosts in Manabí province, Ecuador

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0008286

    Molecular diagnosis of Amphimerus sp. metacercariae based on PCR-RFLP. (A) Multiple metacercariae were isolated from fish collected at each community. (B) Different metacercariae identified based on PCR-RFLP as shown in panel D. (C) First round of PCR amplification using universal trematode primers (green arrow). (D) Digested (L1-L6) and undigested (L7) PCR products by the restriction enzyme Hind III. Lanes 1–5 corresponds to (L1) adult Amphimerus sp. flukes from a human case at Esmeraldas, followed by adult parasites from a human (L2), a dog (L3), and a cat (L4), and (L5) Amphimerus sp. metacercariae from freshwater fish (i.e., Bryconamericus bucay ) from Pedro Pablo Gómez; the two fragments correspond to 374 and 140 base pairs (red arrows). Lane 6 corresponds to the metacercariae of Haplorchis pumilio with fragments of 440 and 86 base pairs (blue arrows). Lane 7 corresponds to unidentified metacercariae from fish in Pedro Pablo Gómez. L: 100 base pairs DNA Ladder (New England Biolabs, Ipswich, Massachusetts, United States).
    Figure Legend Snippet: Molecular diagnosis of Amphimerus sp. metacercariae based on PCR-RFLP. (A) Multiple metacercariae were isolated from fish collected at each community. (B) Different metacercariae identified based on PCR-RFLP as shown in panel D. (C) First round of PCR amplification using universal trematode primers (green arrow). (D) Digested (L1-L6) and undigested (L7) PCR products by the restriction enzyme Hind III. Lanes 1–5 corresponds to (L1) adult Amphimerus sp. flukes from a human case at Esmeraldas, followed by adult parasites from a human (L2), a dog (L3), and a cat (L4), and (L5) Amphimerus sp. metacercariae from freshwater fish (i.e., Bryconamericus bucay ) from Pedro Pablo Gómez; the two fragments correspond to 374 and 140 base pairs (red arrows). Lane 6 corresponds to the metacercariae of Haplorchis pumilio with fragments of 440 and 86 base pairs (blue arrows). Lane 7 corresponds to unidentified metacercariae from fish in Pedro Pablo Gómez. L: 100 base pairs DNA Ladder (New England Biolabs, Ipswich, Massachusetts, United States).

    Techniques Used: Polymerase Chain Reaction, Isolation, Fluorescence In Situ Hybridization, Amplification

    4) Product Images from "GES Extended-Spectrum ?-Lactamases in Acinetobacter baumannii Isolates in Belgium ▿"

    Article Title: GES Extended-Spectrum ?-Lactamases in Acinetobacter baumannii Isolates in Belgium ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00871-10

    Crude plasmid extracts and banding patterns of plasmids pGES-11 and pGES-12 corestricted with HindIII and EcoRI. Lane M, 2 log DNA ladder (0.1 to 10.0 kb); lane 1, pGES-11 (from isolate 9027) extract digested with EcoRI and HindIII; lane 2, pGES-12 (from
    Figure Legend Snippet: Crude plasmid extracts and banding patterns of plasmids pGES-11 and pGES-12 corestricted with HindIII and EcoRI. Lane M, 2 log DNA ladder (0.1 to 10.0 kb); lane 1, pGES-11 (from isolate 9027) extract digested with EcoRI and HindIII; lane 2, pGES-12 (from

    Techniques Used: Plasmid Preparation

    5) Product Images from "Effects of artificially introduced Enterococcus faecalis strains in experimental necrotizing enterocolitis"

    Article Title: Effects of artificially introduced Enterococcus faecalis strains in experimental necrotizing enterocolitis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0216762

    Diversity of E . faecalis in rats. Frequencies of different phenotypes ( A ) and different strains ( B ) within a group of E . faecalis isolates (n = 147) from 4-day old rats. ( C ) Patterns of genomic DNA Hin d III fragments of indicated strains. Note dissimilarity of DNA patterns A (55–249, 49–171, and BB70) and B (265, BB24).
    Figure Legend Snippet: Diversity of E . faecalis in rats. Frequencies of different phenotypes ( A ) and different strains ( B ) within a group of E . faecalis isolates (n = 147) from 4-day old rats. ( C ) Patterns of genomic DNA Hin d III fragments of indicated strains. Note dissimilarity of DNA patterns A (55–249, 49–171, and BB70) and B (265, BB24).

    Techniques Used:

    6) Product Images from "Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis"

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    Journal: eLife

    doi: 10.7554/eLife.19669

    Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from spo11 strains with inserts at HIS4 (top) and at URA3 (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.015
    Figure Legend Snippet: Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from spo11 strains with inserts at HIS4 (top) and at URA3 (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.015

    Techniques Used:

    Spo11-initiated events at the two insert loci. ( A ) Spo11-catalyzed DSBs are more frequent at HIS4 that at URA3 . Left—Southern blots of Eco RI digests of DNA from vde∆ strains, probed with pBR322 sequences, showing Spo11-DSBs in the Parent 2 insert (see Figure 1 ) in resection/repair-deficient sae2∆ mutant strains. Right—location of DSBs and probe and DSB frequencies (average of 7 and 8 hr samples from a single experiment; error bars represent range). Spo11-DSBs in the Parent 1 inserts at HIS4 and URA3 were at different locations within the insert, but displayed similar ratios between the two loci (data not shown). ( B ) Southern blots of Hin dIII digests of DNA from vde∆ strains, to detect total Spo11-initiated crossovers. ( C ) Southern blots of Hin dIII-VDE double digests of the same samples, to determine the background contribution of Spo11-initiated COs in subsequent experiments measuring VDE-initiated COs, which will be VDE-resistant due to conversion of the VRS site to VRS103 . Probes were as shown in Figure 1 . ( D ) Quantification of data in panels B (total COs; filled circles) and C (VDE-resistant COs; open circles). Data are from a single experiment. DOI: http://dx.doi.org/10.7554/eLife.19669.004
    Figure Legend Snippet: Spo11-initiated events at the two insert loci. ( A ) Spo11-catalyzed DSBs are more frequent at HIS4 that at URA3 . Left—Southern blots of Eco RI digests of DNA from vde∆ strains, probed with pBR322 sequences, showing Spo11-DSBs in the Parent 2 insert (see Figure 1 ) in resection/repair-deficient sae2∆ mutant strains. Right—location of DSBs and probe and DSB frequencies (average of 7 and 8 hr samples from a single experiment; error bars represent range). Spo11-DSBs in the Parent 1 inserts at HIS4 and URA3 were at different locations within the insert, but displayed similar ratios between the two loci (data not shown). ( B ) Southern blots of Hin dIII digests of DNA from vde∆ strains, to detect total Spo11-initiated crossovers. ( C ) Southern blots of Hin dIII-VDE double digests of the same samples, to determine the background contribution of Spo11-initiated COs in subsequent experiments measuring VDE-initiated COs, which will be VDE-resistant due to conversion of the VRS site to VRS103 . Probes were as shown in Figure 1 . ( D ) Quantification of data in panels B (total COs; filled circles) and C (VDE-resistant COs; open circles). Data are from a single experiment. DOI: http://dx.doi.org/10.7554/eLife.19669.004

    Techniques Used: Mutagenesis

    70–80% of VDE-DSBs are repaired. ( A ) Fraction of inserts remaining, calculated using Hin dIII digests (see Figure 1 ). For the arg4-VRS103 insert, the ratio (Parent 2 + CO2)/ (0.5 x LC) was calculated at 9 hr, and was then normalized to the 0 hr value. For the arg4-VRS insert, a similar calculation was made: (Parent 1 + NCO + CO1)/(0.5 x LC) ( B ) Relative recovery of interhomolog recombination products, calculated using Hin dIII-VDE double digests (see Figure 1 ). The sum of CO (average of CO1 and CO2) and NCO frequencies was divided by the frequency of total DSBs, as calculated in Figure 2A . Data are the average of two independent experiments; error bars represent range. DOI: http://dx.doi.org/10.7554/eLife.19669.006
    Figure Legend Snippet: 70–80% of VDE-DSBs are repaired. ( A ) Fraction of inserts remaining, calculated using Hin dIII digests (see Figure 1 ). For the arg4-VRS103 insert, the ratio (Parent 2 + CO2)/ (0.5 x LC) was calculated at 9 hr, and was then normalized to the 0 hr value. For the arg4-VRS insert, a similar calculation was made: (Parent 1 + NCO + CO1)/(0.5 x LC) ( B ) Relative recovery of interhomolog recombination products, calculated using Hin dIII-VDE double digests (see Figure 1 ). The sum of CO (average of CO1 and CO2) and NCO frequencies was divided by the frequency of total DSBs, as calculated in Figure 2A . Data are the average of two independent experiments; error bars represent range. DOI: http://dx.doi.org/10.7554/eLife.19669.006

    Techniques Used:

    Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Probes and gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.009
    Figure Legend Snippet: Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Probes and gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.009

    Techniques Used:

    Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. In the gel with Hin DIII digests of samples from a pch2∆ mm4-mn yen1∆ slx1∆ strain with inserts at URA3 , the 9 hr sample was originally loaded between the 4 and 5 hr samples; this image was cut and spliced as indicated by vertical lines for presentation purposes. DOI: http://dx.doi.org/10.7554/eLife.19669.012
    Figure Legend Snippet: Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. In the gel with Hin DIII digests of samples from a pch2∆ mm4-mn yen1∆ slx1∆ strain with inserts at URA3 , the 9 hr sample was originally loaded between the 4 and 5 hr samples; this image was cut and spliced as indicated by vertical lines for presentation purposes. DOI: http://dx.doi.org/10.7554/eLife.19669.012

    Techniques Used:

    7) Product Images from "Emergence of Resistance among USA300 Methicillin-Resistant Staphylococcus aureus Isolates Causing Invasive Disease in the United States ▿"

    Article Title: Emergence of Resistance among USA300 Methicillin-Resistant Staphylococcus aureus Isolates Causing Invasive Disease in the United States ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00351-10

    Gel showing a summary of Southern hybridization data from six gels of HindIII-restricted RN4220-RF transconjugant plasmid DNA, pSK41 (lane 9), and pUSA03 and pUSA02 (lane 16). Four probes each hybridized with a unique HindIII fragment in all plasmid DNA
    Figure Legend Snippet: Gel showing a summary of Southern hybridization data from six gels of HindIII-restricted RN4220-RF transconjugant plasmid DNA, pSK41 (lane 9), and pUSA03 and pUSA02 (lane 16). Four probes each hybridized with a unique HindIII fragment in all plasmid DNA

    Techniques Used: Hybridization, Plasmid Preparation

    8) Product Images from "Genomic Analysis Reveals Mycoplasma pneumoniae Repetitive Element 1-Mediated Recombination in a Clinical Isolate "

    Article Title: Genomic Analysis Reveals Mycoplasma pneumoniae Repetitive Element 1-Mediated Recombination in a Clinical Isolate

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01621-07

    Southern analysis of strains M129 and S1. Chromosomal DNA isolated from M129 (lanes 1, 3, 5, and 7) and S1 (lanes 2, 4, 6, and 8) was digested to completion with EcoRI and HindIII, and the fragments generated were separated on 1% agarose gels
    Figure Legend Snippet: Southern analysis of strains M129 and S1. Chromosomal DNA isolated from M129 (lanes 1, 3, 5, and 7) and S1 (lanes 2, 4, 6, and 8) was digested to completion with EcoRI and HindIII, and the fragments generated were separated on 1% agarose gels

    Techniques Used: Isolation, Generated

    9) Product Images from "Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ"

    Article Title: Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

    Journal: BMC Genomics

    doi: 10.1186/s12864-016-3331-9

    Generation of floxed mice by the enhanced PITCh system. a Targeting strategy for the generation of flox Col12a1 mice by the enhanced PITCh system. Purple highlights indicate microhomologies between endogenous Col12a1 locus and PITCh-donor. Blue characters indicate CRISPR target sequences. Red characters indicate protospacer adjacent motif (PAM) sequences. Yellow lightnings indicate DSB sites. b Schematic diagram of pronuclear injection of Cas9 protein, Col12a1 -left, -right, and gRNA-s1 crRNAs, tracrRNA, PITCh-donor, and Exo1 mRNA. The red, purple, and blue boxes indicate the insert, Col12a1 microhomologies, and gRNA-s1 target sequences, respectively. c PCR screenings of newborns. d PCR-RFLP (restriction fragment length polymorphism) screenings of floxed newborn mice. e Summary of flox Col12a1 mouse production by the enhanced PITCh system. f Sequences of boundaries between Col12a1 and LoxPs. Blue, green, and red characters indicate microhomologies, HindIII sites, and LoxPs, respectively. g in vitro Cre-recombination assay. Cloned PCR products of flox alleles from three flox Col12a1 mice and genomic PCR of wildtype were incubated with or without Cre-recombinase. LF: left forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, and WT: wildtype
    Figure Legend Snippet: Generation of floxed mice by the enhanced PITCh system. a Targeting strategy for the generation of flox Col12a1 mice by the enhanced PITCh system. Purple highlights indicate microhomologies between endogenous Col12a1 locus and PITCh-donor. Blue characters indicate CRISPR target sequences. Red characters indicate protospacer adjacent motif (PAM) sequences. Yellow lightnings indicate DSB sites. b Schematic diagram of pronuclear injection of Cas9 protein, Col12a1 -left, -right, and gRNA-s1 crRNAs, tracrRNA, PITCh-donor, and Exo1 mRNA. The red, purple, and blue boxes indicate the insert, Col12a1 microhomologies, and gRNA-s1 target sequences, respectively. c PCR screenings of newborns. d PCR-RFLP (restriction fragment length polymorphism) screenings of floxed newborn mice. e Summary of flox Col12a1 mouse production by the enhanced PITCh system. f Sequences of boundaries between Col12a1 and LoxPs. Blue, green, and red characters indicate microhomologies, HindIII sites, and LoxPs, respectively. g in vitro Cre-recombination assay. Cloned PCR products of flox alleles from three flox Col12a1 mice and genomic PCR of wildtype were incubated with or without Cre-recombinase. LF: left forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, and WT: wildtype

    Techniques Used: Mouse Assay, CRISPR, Injection, Polymerase Chain Reaction, In Vitro, Recombination Assay, Clone Assay, Incubation, Marker

    10) Product Images from "Single-stranded DNA and RNA origami"

    Article Title: Single-stranded DNA and RNA origami

    Journal: Science (New York, N.Y.)

    doi: 10.1126/science.aao2648

    Schematic of ssOrigami synthesis and replication by in vitro PCR and by in vivo cloning of ssOrigami genes. ( A ) One-step PCR with two double-stranded gBlock templates containing 30-bp sequence overlap (yellow sections) and two modified primers (phosphorothioate modification on green primer and phosphorylation modification on red primer). ( B ) Double-stranded PCR product with modified 5′ ends. (C) ssDNA product after lambda exonuclease digestion. Phosphorothioate modification protects the forward strand from being digested. ( D ) Folded ssOrigami structure. Note that the folded ssOrigami product can be directly used as a template for its PCR replication. ( E ) Double-stranded gBlock DNA fragments with restriction enzyme sites designed at both ends. ( F ) Ligation of two half fragments into linearized pGEM-7zf (−) vector to form the full-length ssOrigami gene. ( G ) The ligation products were transformed into E. coli NEB stable competent cells. ( H ) Full-length ssOrigami genes were amplified as plasmid DNA in E. coli NEB stable cells. ( I ) The harvested genes were treated by the nicking endonuclease Nb.BbvCI and the restriction endonuclease Hind III. ( J . ( K and L ) Schematic (K) and AFM images [(K), zoomed-in; (L), large field of view] of the 5 × 5 ssOrigami structures produced by the PCR synthesis [first cycle in (A) to (D)]. ( M ) AFM image of 5 × 5 ssOrigami structures produced by PCR replication method [the second cycle in (A) to (D), that is, the re-PCR product]. ( N ) AFM image of 5 × 5 rhombus ssOrigami produced by in vivo cloning method. Detailed experimental information is shown in sections S6 (in vitro PCR) and S7 (in vivo cloning).
    Figure Legend Snippet: Schematic of ssOrigami synthesis and replication by in vitro PCR and by in vivo cloning of ssOrigami genes. ( A ) One-step PCR with two double-stranded gBlock templates containing 30-bp sequence overlap (yellow sections) and two modified primers (phosphorothioate modification on green primer and phosphorylation modification on red primer). ( B ) Double-stranded PCR product with modified 5′ ends. (C) ssDNA product after lambda exonuclease digestion. Phosphorothioate modification protects the forward strand from being digested. ( D ) Folded ssOrigami structure. Note that the folded ssOrigami product can be directly used as a template for its PCR replication. ( E ) Double-stranded gBlock DNA fragments with restriction enzyme sites designed at both ends. ( F ) Ligation of two half fragments into linearized pGEM-7zf (−) vector to form the full-length ssOrigami gene. ( G ) The ligation products were transformed into E. coli NEB stable competent cells. ( H ) Full-length ssOrigami genes were amplified as plasmid DNA in E. coli NEB stable cells. ( I ) The harvested genes were treated by the nicking endonuclease Nb.BbvCI and the restriction endonuclease Hind III. ( J . ( K and L ) Schematic (K) and AFM images [(K), zoomed-in; (L), large field of view] of the 5 × 5 ssOrigami structures produced by the PCR synthesis [first cycle in (A) to (D)]. ( M ) AFM image of 5 × 5 ssOrigami structures produced by PCR replication method [the second cycle in (A) to (D), that is, the re-PCR product]. ( N ) AFM image of 5 × 5 rhombus ssOrigami produced by in vivo cloning method. Detailed experimental information is shown in sections S6 (in vitro PCR) and S7 (in vivo cloning).

    Techniques Used: In Vitro, Polymerase Chain Reaction, In Vivo, Clone Assay, Sequencing, Modification, Ligation, Plasmid Preparation, Transformation Assay, Amplification, Produced

    11) Product Images from "Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub"

    Article Title: Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt067

    Visualization of nuclear compartments and chromatin domains in non-treated liver cells ( A ) and the same cells treated according to the 3C protocol up to the ligation step ( B ). The insoluble fraction was collected after HindIII digestion and 1.6% SDS extraction. (a–e) Immunostaining with antibodies against nucleolin (a), Sc35 (b), DNA topoisomerase II (c), H3K9me3 (d) and H3K27me3 (e). (f) Visualization of the chromosome 7 territory (FISH with a library of the chromosome 7–specific probes). In both sections of the Figure, the results of immunostaining are shown in the first row (red) and counterstaining of DNA with DAPI is shown in the second row (blue). The superimposition of the immunostaining and counterstaining of DNA is shown in the third row. Scale bar: 5 µm.
    Figure Legend Snippet: Visualization of nuclear compartments and chromatin domains in non-treated liver cells ( A ) and the same cells treated according to the 3C protocol up to the ligation step ( B ). The insoluble fraction was collected after HindIII digestion and 1.6% SDS extraction. (a–e) Immunostaining with antibodies against nucleolin (a), Sc35 (b), DNA topoisomerase II (c), H3K9me3 (d) and H3K27me3 (e). (f) Visualization of the chromosome 7 territory (FISH with a library of the chromosome 7–specific probes). In both sections of the Figure, the results of immunostaining are shown in the first row (red) and counterstaining of DNA with DAPI is shown in the second row (blue). The superimposition of the immunostaining and counterstaining of DNA is shown in the third row. Scale bar: 5 µm.

    Techniques Used: Ligation, Immunostaining, Fluorescence In Situ Hybridization

    Electron microscopic analysis of the insoluble 3C material from liver cells at different steps of the 3C procedure. After formaldehyde cross-linking ( A and A’ ), after isolation of nuclei and extraction with 0.3% SDS followed by 1.8% Triton X-100 ( B and B’ ) and after digestion with HindIII restriction endonuclease followed by extraction with 1.6% SDS ( C and C’ ). Panels below show the enlarged framed region of the above images. Scale bars: 1 µm (A–C) and 250 nm (A’–C’).
    Figure Legend Snippet: Electron microscopic analysis of the insoluble 3C material from liver cells at different steps of the 3C procedure. After formaldehyde cross-linking ( A and A’ ), after isolation of nuclei and extraction with 0.3% SDS followed by 1.8% Triton X-100 ( B and B’ ) and after digestion with HindIII restriction endonuclease followed by extraction with 1.6% SDS ( C and C’ ). Panels below show the enlarged framed region of the above images. Scale bars: 1 µm (A–C) and 250 nm (A’–C’).

    Techniques Used: Isolation

    Frequencies of ligation of the fragment harboring the Hbb-b1 promoter with several selected fragments of the β-globin gene domain in soluble and insoluble portions of the 3C material. ( A ) Results of standard 3C analysis performed without fractionating the 3C material. ( B ) Results of 3C analysis performed separately on soluble (super) and insoluble (debris) fractions. ( C ) The same as (B) after normalization of the ligation frequencies to the amount of DNA in the samples. ( D ) The same as (C), soluble fraction only. On the top of each graph, a map of the domain is shown, with β-globin genes, olfactory receptor genes and DNase I hypersensitive sites shown by red arrows, blue arrows and black vertical lines, respectively. Plotted on the horizontal axis are the fragment positions. The scale is in kilobases, and according to GenBank entry NT_039433, the ‘0’ point corresponds to the start of the Hbb-y gene. The black rectangle in the background of each graph shows the anchor fragment, and the gray rectangles indicate test fragments. Plotted on the vertical axis are the ligation frequencies; the highest ligation frequency observed is set to 100 [the frequency of ligation between the anchor fragment and the upstream restriction fragment in the total 3C material from fetal liver cells (A) or in the insoluble portion of the 3C material from fetal liver cells (B and C) or the soluble portion of the 3C material from fetal brain cells (D)]. Red and blue lines show the results for liver and brain cells, respectively; solid lines show the results for the total 3C material (A) or the insoluble portion of the 3C material (B and C); dotted lines show the results for the soluble portion of the 3C material. Ligation frequencies of HindIII and MboI fragments are presented on the left and the right graphs, respectively. The error bars represent SEM for three independent experiments.
    Figure Legend Snippet: Frequencies of ligation of the fragment harboring the Hbb-b1 promoter with several selected fragments of the β-globin gene domain in soluble and insoluble portions of the 3C material. ( A ) Results of standard 3C analysis performed without fractionating the 3C material. ( B ) Results of 3C analysis performed separately on soluble (super) and insoluble (debris) fractions. ( C ) The same as (B) after normalization of the ligation frequencies to the amount of DNA in the samples. ( D ) The same as (C), soluble fraction only. On the top of each graph, a map of the domain is shown, with β-globin genes, olfactory receptor genes and DNase I hypersensitive sites shown by red arrows, blue arrows and black vertical lines, respectively. Plotted on the horizontal axis are the fragment positions. The scale is in kilobases, and according to GenBank entry NT_039433, the ‘0’ point corresponds to the start of the Hbb-y gene. The black rectangle in the background of each graph shows the anchor fragment, and the gray rectangles indicate test fragments. Plotted on the vertical axis are the ligation frequencies; the highest ligation frequency observed is set to 100 [the frequency of ligation between the anchor fragment and the upstream restriction fragment in the total 3C material from fetal liver cells (A) or in the insoluble portion of the 3C material from fetal liver cells (B and C) or the soluble portion of the 3C material from fetal brain cells (D)]. Red and blue lines show the results for liver and brain cells, respectively; solid lines show the results for the total 3C material (A) or the insoluble portion of the 3C material (B and C); dotted lines show the results for the soluble portion of the 3C material. Ligation frequencies of HindIII and MboI fragments are presented on the left and the right graphs, respectively. The error bars represent SEM for three independent experiments.

    Techniques Used: Ligation

    12) Product Images from "A cell cycle-dependent CRISPR-Cas9 activation system based on an anti-CRISPR protein shows improved genome editing accuracy"

    Article Title: A cell cycle-dependent CRISPR-Cas9 activation system based on an anti-CRISPR protein shows improved genome editing accuracy

    Journal: Communications Biology

    doi: 10.1038/s42003-020-01340-2

    Genome editing through HDR by using repair template ssODN. a Method to confirm the HDR efficiency using HindIII restriction enzyme. b – d Results of specific HDR and off-target mutation at three different targets ( b : AAVS1 gene, c : EMX1 gene, d : VEGFA gene). Each PCR product amplified from extracted genomic DNA reacted with HindIII for target HDR or T7E1 for target T7E1 and off-target mutation. Target T7E1 includes mutagenesis by HDR and NHEJ. The editing efficiency was calculated by the formula; 100 × ((b + c)/(a + b + c)) for HDR, 100 × (1–sqrt(1 – (b + c)/(a + b + c))) for off-target mutation, where “a” is the integrated intensity of the undigested PCR product, and “b” and “c” are the integrated intensities of each cleavage product. n = 6 for SpyCas9 and AcrIIA4-Cdt1-2A-Cas9 samples. n = 3 for AcrIIA4-2A-Cas9 sample. Actual electrophoresis images are shown in Supplementary Fig. 6 . Significance in difference was tested by Student’s t -test.
    Figure Legend Snippet: Genome editing through HDR by using repair template ssODN. a Method to confirm the HDR efficiency using HindIII restriction enzyme. b – d Results of specific HDR and off-target mutation at three different targets ( b : AAVS1 gene, c : EMX1 gene, d : VEGFA gene). Each PCR product amplified from extracted genomic DNA reacted with HindIII for target HDR or T7E1 for target T7E1 and off-target mutation. Target T7E1 includes mutagenesis by HDR and NHEJ. The editing efficiency was calculated by the formula; 100 × ((b + c)/(a + b + c)) for HDR, 100 × (1–sqrt(1 – (b + c)/(a + b + c))) for off-target mutation, where “a” is the integrated intensity of the undigested PCR product, and “b” and “c” are the integrated intensities of each cleavage product. n = 6 for SpyCas9 and AcrIIA4-Cdt1-2A-Cas9 samples. n = 3 for AcrIIA4-2A-Cas9 sample. Actual electrophoresis images are shown in Supplementary Fig. 6 . Significance in difference was tested by Student’s t -test.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Non-Homologous End Joining, Electrophoresis

    Genome editing using truncated sgRNA. a Truncated sgRNA whose target sequence is shorter than the normal one. b,c Results of target HDR and off-target mutation at two different targets ( b : EMX1 gene, c : VEGFA gene). Each PCR product amplified from extracted genomic DNA reacted with HindIII for HDR or T7E1 for off-target mutation. The editing efficiency was calculated by the formula; 100 × ((b + c)/(a + b + c)) for HDR, 100 × (1–sqrt(1 – (b + c)/(a + b + c))) for off-target mutation, where “a” is the integrated intensity of the undigested PCR product, and “b” and “c” are the integrated intensities of each cleavage product. n = 3 for EMX1 samples and VEGFA SpyCas9 sample. n = 5 for VEGFA AcrIIA4-Cdt1-2A-Cas9 sample. Actual electrophoresis images are shown in Supplementary Fig. 6 . Significance in difference was tested by Student’s t-test.
    Figure Legend Snippet: Genome editing using truncated sgRNA. a Truncated sgRNA whose target sequence is shorter than the normal one. b,c Results of target HDR and off-target mutation at two different targets ( b : EMX1 gene, c : VEGFA gene). Each PCR product amplified from extracted genomic DNA reacted with HindIII for HDR or T7E1 for off-target mutation. The editing efficiency was calculated by the formula; 100 × ((b + c)/(a + b + c)) for HDR, 100 × (1–sqrt(1 – (b + c)/(a + b + c))) for off-target mutation, where “a” is the integrated intensity of the undigested PCR product, and “b” and “c” are the integrated intensities of each cleavage product. n = 3 for EMX1 samples and VEGFA SpyCas9 sample. n = 5 for VEGFA AcrIIA4-Cdt1-2A-Cas9 sample. Actual electrophoresis images are shown in Supplementary Fig. 6 . Significance in difference was tested by Student’s t-test.

    Techniques Used: Sequencing, Mutagenesis, Polymerase Chain Reaction, Amplification, Electrophoresis

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202138

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

    Techniques Used: Electrophoresis, Sequencing

    14) Product Images from "DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats"

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats

    Journal: bioRxiv

    doi: 10.1101/2020.06.20.162743

    Pooled measurement of DNA polymerase stalling at STRs. (a) Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structures annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. (b) Extended and stalled products were then analysed by denaturing Poly Acrylamide Gel (PAGE) electrophoresis, recovered from the gel matrix and prepared for high throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence form the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, are reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear overtime.
    Figure Legend Snippet: Pooled measurement of DNA polymerase stalling at STRs. (a) Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structures annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. (b) Extended and stalled products were then analysed by denaturing Poly Acrylamide Gel (PAGE) electrophoresis, recovered from the gel matrix and prepared for high throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence form the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, are reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear overtime.

    Techniques Used: High Throughput Screening Assay, Primer Extension Assay, DNA Synthesis, Microarray, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Produced, Acrylamide Gel Assay, Polyacrylamide Gel Electrophoresis, Electrophoresis, Next-Generation Sequencing, Sequencing, Fluorescence, Imaging

    15) Product Images from "The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness"

    Article Title: The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness

    Journal: Viruses

    doi: 10.3390/v10070343

    MotB and H-NS bind to both unmodified λ and GHme-C modified T4 DNA. Agarose gel shows the DNA (500 ng) λ or T4 DNA pretreated with HindIII and SspI restriction nucleases (lanes 1 and 5, respectively), after incubation with 60 pmol MotB (lanes 2 and 6), 60 pmol H-NS (lanes 3 and 7), or both (lanes 4 and 8). DNA was visualized by ethidium bromide staining and UV illumination.
    Figure Legend Snippet: MotB and H-NS bind to both unmodified λ and GHme-C modified T4 DNA. Agarose gel shows the DNA (500 ng) λ or T4 DNA pretreated with HindIII and SspI restriction nucleases (lanes 1 and 5, respectively), after incubation with 60 pmol MotB (lanes 2 and 6), 60 pmol H-NS (lanes 3 and 7), or both (lanes 4 and 8). DNA was visualized by ethidium bromide staining and UV illumination.

    Techniques Used: Modification, Agarose Gel Electrophoresis, Incubation, Staining

    16) Product Images from "Characterisation of a novel ATR-dependent, Chk1-independent, intra-S phase checkpoint that suppresses initiation of replication in Xenopus"

    Article Title: Characterisation of a novel ATR-dependent, Chk1-independent, intra-S phase checkpoint that suppresses initiation of replication in Xenopus

    Journal: Journal of cell science

    doi: 10.1242/jcs.01400

    Inhibition of CDK activity prevents later origins from firing. ( A ) Sperm nuclei were incubated at 15 ng DNA/μl in egg extract supplemented with [α- 32 P]dATP. Aliquots were supplemented with 0.5 mM roscovitine at the following times: filled triangles, 20 min; filled diamonds, 25 min; filled circles 30 min; open triangles, 35 min; open diamonds, 40 min; open circles, 50 min. Samples with no added roscovitine are shown by open squares. At the indicated times, samples were assayed for total DNA synthesis. ( B ) Sperm nuclei were incubated at 15 ng DNA/μl in egg extract supplemented with biotin-dUTP. 0.5 mM roscovitine was added at the indicated times. At 120 min, nuclei were isolated and stained with Texas Red streptavidin to reveal nuclei which had undergone DNA replication. The percentage of biotin-positive nuclei for each point is shown. ( C, D ) Sperm nuclei were incubated at 15 ng DNA/μl in egg extract. At 35 minutes (C) or 45 minutes (D), aliquots were supplemented with 0.5mM roscovitine. Samples were pulse-labelled with [α- 32 P]dATP for 2 minutes at the indicated times. DNA was separated on an alkaline agarose gel and autoradiographed. The migration of end-labelled λ-HindIII DNA is also shown.
    Figure Legend Snippet: Inhibition of CDK activity prevents later origins from firing. ( A ) Sperm nuclei were incubated at 15 ng DNA/μl in egg extract supplemented with [α- 32 P]dATP. Aliquots were supplemented with 0.5 mM roscovitine at the following times: filled triangles, 20 min; filled diamonds, 25 min; filled circles 30 min; open triangles, 35 min; open diamonds, 40 min; open circles, 50 min. Samples with no added roscovitine are shown by open squares. At the indicated times, samples were assayed for total DNA synthesis. ( B ) Sperm nuclei were incubated at 15 ng DNA/μl in egg extract supplemented with biotin-dUTP. 0.5 mM roscovitine was added at the indicated times. At 120 min, nuclei were isolated and stained with Texas Red streptavidin to reveal nuclei which had undergone DNA replication. The percentage of biotin-positive nuclei for each point is shown. ( C, D ) Sperm nuclei were incubated at 15 ng DNA/μl in egg extract. At 35 minutes (C) or 45 minutes (D), aliquots were supplemented with 0.5mM roscovitine. Samples were pulse-labelled with [α- 32 P]dATP for 2 minutes at the indicated times. DNA was separated on an alkaline agarose gel and autoradiographed. The migration of end-labelled λ-HindIII DNA is also shown.

    Techniques Used: Inhibition, Activity Assay, Incubation, DNA Synthesis, Isolation, Staining, Agarose Gel Electrophoresis, Migration

    Aphidicolin induces a caffeine-sensitive replication checkpoint. ( A - D ) Sperm nuclei were incubated at 15 ng DNA/μl in interphase egg extract supplemented with [α- 32 P]dATP plus various concentrations of aphidicolin plus or minus 5 mM caffeine. (A) Total DNA synthesis was measured at 120 min. (B) DNA synthesis was measured between 15 and 120 minutes as indicated. (C, D) At 120 min, DNA was separated on an alkaline agarose gel and autoradiographed. The migration of end-labelled λ-HindIII DNA is also shown. ( E , F ) Sperm nuclei were incubated at 15 ng DNA/μl for 35 minutes in interphase egg extract supplemented with 7.5μM aphidicolin. The reaction was split in two, and supplemented with 0.5 mM roscovitine minus (E) or plus (F) 5 mM caffeine. At 5 minute intervals, aliquots were pulse-labelled for 2 minutes with [α- 32 P]dATP, then DNA was separated on an alkaline agarose gel and autoradiographed. The migration of end-labelled λ-HindIII DNA is also shown.
    Figure Legend Snippet: Aphidicolin induces a caffeine-sensitive replication checkpoint. ( A - D ) Sperm nuclei were incubated at 15 ng DNA/μl in interphase egg extract supplemented with [α- 32 P]dATP plus various concentrations of aphidicolin plus or minus 5 mM caffeine. (A) Total DNA synthesis was measured at 120 min. (B) DNA synthesis was measured between 15 and 120 minutes as indicated. (C, D) At 120 min, DNA was separated on an alkaline agarose gel and autoradiographed. The migration of end-labelled λ-HindIII DNA is also shown. ( E , F ) Sperm nuclei were incubated at 15 ng DNA/μl for 35 minutes in interphase egg extract supplemented with 7.5μM aphidicolin. The reaction was split in two, and supplemented with 0.5 mM roscovitine minus (E) or plus (F) 5 mM caffeine. At 5 minute intervals, aliquots were pulse-labelled for 2 minutes with [α- 32 P]dATP, then DNA was separated on an alkaline agarose gel and autoradiographed. The migration of end-labelled λ-HindIII DNA is also shown.

    Techniques Used: Incubation, DNA Synthesis, Agarose Gel Electrophoresis, Migration

    Fork stability is not significantly affected by the intra-S phase checkpoint. ( A , B , C ) Sperm nuclei were incubated at 10 ng DNA/μl in Xenopus extract; after 35 minutes (early S-phase) extract was supplemented with 40 μM aphidicolin and 0.5 mM roscovitine minus (B) or plus (C) 5 mM caffeine. 0, 10, 25 or 55 minutes after this, nuclei were isolated and transferred to fresh extract supplemented with 0.5 mM roscovitine and [α- 32 P]dATP. Total DNA synthesis was measured at different times after transfer. A schematic outline of the experiment is shown in (A). ( D ) Sperm nuclei were incubated at 10 ng DNA/μl in extract. At 35 min, the extract was supplemented with 7.5 μM aphidicolin and 5 mM caffeine, minus or plus 0.5 mM roscovitine. At the indicated times, samples were pulse-labelled with [α- 32 P]dATP for 2 min, the DNA was isolated and analysed by agarose electrophoresis and autoradiography. The migration of end-labelled λ-HindIII DNA is also shown.
    Figure Legend Snippet: Fork stability is not significantly affected by the intra-S phase checkpoint. ( A , B , C ) Sperm nuclei were incubated at 10 ng DNA/μl in Xenopus extract; after 35 minutes (early S-phase) extract was supplemented with 40 μM aphidicolin and 0.5 mM roscovitine minus (B) or plus (C) 5 mM caffeine. 0, 10, 25 or 55 minutes after this, nuclei were isolated and transferred to fresh extract supplemented with 0.5 mM roscovitine and [α- 32 P]dATP. Total DNA synthesis was measured at different times after transfer. A schematic outline of the experiment is shown in (A). ( D ) Sperm nuclei were incubated at 10 ng DNA/μl in extract. At 35 min, the extract was supplemented with 7.5 μM aphidicolin and 5 mM caffeine, minus or plus 0.5 mM roscovitine. At the indicated times, samples were pulse-labelled with [α- 32 P]dATP for 2 min, the DNA was isolated and analysed by agarose electrophoresis and autoradiography. The migration of end-labelled λ-HindIII DNA is also shown.

    Techniques Used: Incubation, Isolation, DNA Synthesis, Electrophoresis, Autoradiography, Migration

    17) Product Images from "Analyses of germline variants associated with ovarian cancer survival identify functional candidates at the 1q22 and 19p12 outcome loci"

    Article Title: Analyses of germline variants associated with ovarian cancer survival identify functional candidates at the 1q22 and 19p12 outcome loci

    Journal: Oncotarget

    doi: 10.18632/oncotarget.18501

    Two regions containing candidate all chemo PFS candidate variants interact with the ZNF100 promoter in ovarian cancer cell lines at the 19p12 outcome locus The figure shows 3C analyses of interactions between HindIII fragments and the ZNF100 promoter region (highlighted in pink) in COV362 and OVCAR8 cells. For each cell line, interaction frequencies were normalised to those of the fragment proximal to the promoter. Interaction frequencies from three independent biological replicates are shown (error bars represent standard error of the mean). The original variant associated with any chemotherapy PFS, rs3795247, is shown with correlated candidate outcome variants ( r 2 > 0.4) in black. Roadmap Consortium chromatin state segmentation for normal ovarian tissue using a Hidden Markov Model (Chrom HMM) is shown (red = active transcription start sites, dark green = weak transcription, green = strong transcription, green/yellow = genic enhancers, yellow = enhancers, aquamarine = ZNF gene repeats and turquoise = heterochromatin). H3K4Me1 modification in normal ovarian tissue is also indicated. Putative Regulatory Elements (PREs), and their coincident candidate variants, cloned into reporter gene constructs are highlighted in blue.
    Figure Legend Snippet: Two regions containing candidate all chemo PFS candidate variants interact with the ZNF100 promoter in ovarian cancer cell lines at the 19p12 outcome locus The figure shows 3C analyses of interactions between HindIII fragments and the ZNF100 promoter region (highlighted in pink) in COV362 and OVCAR8 cells. For each cell line, interaction frequencies were normalised to those of the fragment proximal to the promoter. Interaction frequencies from three independent biological replicates are shown (error bars represent standard error of the mean). The original variant associated with any chemotherapy PFS, rs3795247, is shown with correlated candidate outcome variants ( r 2 > 0.4) in black. Roadmap Consortium chromatin state segmentation for normal ovarian tissue using a Hidden Markov Model (Chrom HMM) is shown (red = active transcription start sites, dark green = weak transcription, green = strong transcription, green/yellow = genic enhancers, yellow = enhancers, aquamarine = ZNF gene repeats and turquoise = heterochromatin). H3K4Me1 modification in normal ovarian tissue is also indicated. Putative Regulatory Elements (PREs), and their coincident candidate variants, cloned into reporter gene constructs are highlighted in blue.

    Techniques Used: Variant Assay, Modification, Clone Assay, Construct

    18) Product Images from "A nematode effector protein similar to annexins in host plants"

    Article Title: A nematode effector protein similar to annexins in host plants

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erp293

    Genomic DNA of Heterodera schachtii and H. glycines digested with Bam HI (B) and Hin dIII (H) was hybridized on blots with a 162 bp Hg4F01 DIG-labelled cDNA probe and revealed two potential 4F01 annexin-like family members in each cyst nematode species. M, molecular weight marker shown in Kb.
    Figure Legend Snippet: Genomic DNA of Heterodera schachtii and H. glycines digested with Bam HI (B) and Hin dIII (H) was hybridized on blots with a 162 bp Hg4F01 DIG-labelled cDNA probe and revealed two potential 4F01 annexin-like family members in each cyst nematode species. M, molecular weight marker shown in Kb.

    Techniques Used: Molecular Weight, Marker

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

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

    Journal: BioMed Research International

    doi: 10.1155/2014/363790

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

    Techniques Used: Lambda DNA Preparation, Marker

    20) Product Images from "Productive Replication of Human Papillomavirus 31 Requires DNA Repair Factor Nbs1"

    Article Title: Productive Replication of Human Papillomavirus 31 Requires DNA Repair Factor Nbs1

    Journal: Journal of Virology

    doi: 10.1128/JVI.00517-14

    Nbs1 knockdown disrupts MRN complex formation. (A) Whole-cell lysates were harvested from HFK, CIN612, and CIN612 9E cells stably expressing shScramble or shNBS1 at T 0 and after 72 h of differentiation in high-calcium medium (Ca). Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. GAPDH was used as a loading control. (B) Total, nuclear (Nuc), and cytoplasmic (Cyto) lysates were harvested from HFK and CIN612 cells. Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. (C) Total, nuclear (Nuc), and cytoplasmic (Cyto) lysates were harvested from stable CIN612 9E shScramble and CIN612 9E shNBS1 cells. Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. For panels B and C, lamin A/C and tubulin were used to confirm nuclear and cytoplasmic fractionation, respectively. (D) DNA was harvested from CIN612 9E cells at T 0 and after 72 h of differentiation in high-calcium medium with dimethyl sulfoxide (DMSO) as a vehicle control or 50 μM Mre11 inhibitor Mirin. Southern blot analysis was performed to analyze viral genome amplification of DNA digested with BamHI (nonviral genome cutter; upper panel) or HindIII (cuts viral genome once; lower panel). All results are representative of observations of two or more independent experiments.
    Figure Legend Snippet: Nbs1 knockdown disrupts MRN complex formation. (A) Whole-cell lysates were harvested from HFK, CIN612, and CIN612 9E cells stably expressing shScramble or shNBS1 at T 0 and after 72 h of differentiation in high-calcium medium (Ca). Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. GAPDH was used as a loading control. (B) Total, nuclear (Nuc), and cytoplasmic (Cyto) lysates were harvested from HFK and CIN612 cells. Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. (C) Total, nuclear (Nuc), and cytoplasmic (Cyto) lysates were harvested from stable CIN612 9E shScramble and CIN612 9E shNBS1 cells. Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. For panels B and C, lamin A/C and tubulin were used to confirm nuclear and cytoplasmic fractionation, respectively. (D) DNA was harvested from CIN612 9E cells at T 0 and after 72 h of differentiation in high-calcium medium with dimethyl sulfoxide (DMSO) as a vehicle control or 50 μM Mre11 inhibitor Mirin. Southern blot analysis was performed to analyze viral genome amplification of DNA digested with BamHI (nonviral genome cutter; upper panel) or HindIII (cuts viral genome once; lower panel). All results are representative of observations of two or more independent experiments.

    Techniques Used: Stable Transfection, Expressing, Fractionation, Southern Blot, Amplification

    Phosphorylation of ATM and Chk2 is maintained with Nbs1 knockdown upon differentiation. (A) Whole-cell lysates were harvested from HFKs and CIN612 9E cells, as well as CIN612 9E cells stably expressing shScramble or shNBS1 cells at T 0 and 72 h after differentiation in high-calcium medium. Immunoblotting was performed using antibodies to phosphorylated ATM (Ser1981) (pATM), total ATM, and Nbs1. Tubulin was used as a loading control. Protein levels were quantified using ImageJ, with phosphorylated protein levels normalized first to total levels and then to tubulin. Levels for this representative experiment are graphed as fold change compared to the T 0 HFK sample, which is set to 1. (B) Whole-cell lysates were harvested from HFK, CIN612 9E, CIN612 9E shScramble, and CIN612 9E shNBS1 cells at T 0 and 72 h after differentiation in high-calcium medium. Immunoblotting was performed using antibodies to phosphorylated Chk2 (Thr68) (pChk2), total Chk2, and Nbs1. GAPDH was used as a loading control. Protein levels were quantified using ImageJ as indicated above. Shown is a representative experiment where levels are graphed as fold change compared to the T 0 HFK sample, which is set at 1. (C) DNA was harvested from CIN612 9E, CIN612 9E shScramble, and CIN612 9E shNBS1 cells at T 0 and after 72 h of differentiation in high-calcium medium and linearized by digestion with HindIII. HPV episomes were visualized via Southern blot analysis. Results shown are representative observations of four or more independent experiments.
    Figure Legend Snippet: Phosphorylation of ATM and Chk2 is maintained with Nbs1 knockdown upon differentiation. (A) Whole-cell lysates were harvested from HFKs and CIN612 9E cells, as well as CIN612 9E cells stably expressing shScramble or shNBS1 cells at T 0 and 72 h after differentiation in high-calcium medium. Immunoblotting was performed using antibodies to phosphorylated ATM (Ser1981) (pATM), total ATM, and Nbs1. Tubulin was used as a loading control. Protein levels were quantified using ImageJ, with phosphorylated protein levels normalized first to total levels and then to tubulin. Levels for this representative experiment are graphed as fold change compared to the T 0 HFK sample, which is set to 1. (B) Whole-cell lysates were harvested from HFK, CIN612 9E, CIN612 9E shScramble, and CIN612 9E shNBS1 cells at T 0 and 72 h after differentiation in high-calcium medium. Immunoblotting was performed using antibodies to phosphorylated Chk2 (Thr68) (pChk2), total Chk2, and Nbs1. GAPDH was used as a loading control. Protein levels were quantified using ImageJ as indicated above. Shown is a representative experiment where levels are graphed as fold change compared to the T 0 HFK sample, which is set at 1. (C) DNA was harvested from CIN612 9E, CIN612 9E shScramble, and CIN612 9E shNBS1 cells at T 0 and after 72 h of differentiation in high-calcium medium and linearized by digestion with HindIII. HPV episomes were visualized via Southern blot analysis. Results shown are representative observations of four or more independent experiments.

    Techniques Used: Stable Transfection, Expressing, Southern Blot

    Nbs1 is necessary for productive viral replication. (A) DNA was harvested from CIN612 9E cells stably expressing a Scramble shRNA or Nbs1 shRNA at T 0 (undifferentiated) or after 48 and 96 h of differentiation in high-calcium medium. Southern blot analysis was performed to analyze viral genome amplification. The bar graph represents quantification of the episome copy number present at each time point, relative to T 0 shScramble, which was set to 1. Densitometry was performed using ImageJ. Ca, calcium. (B) Total protein was harvested from CIN612 9E shScramble and shNbs1 cells at T 0 or after 48 and 96 h of differentiation in high-calcium medium. Western blot analysis was performed using antibodies to Nbs1 and involucrin, and GAPDH as a loading control. (C) DNA was harvested from human foreskin keratinocytes stably maintaining HPV31 genomes (HFK-31) at T 0 or after 24 and 48 h of differentiation in methylcellulose (MC) and analyzed by Southern blotting for amplification of viral genomes. DNA samples were digested with BamHI (does not cut the viral genome; upper panel) or with HindIII to linearize viral genomes. The bar graph represents quantification of the episome copy number present at each time point, relative to T 0 shScramble (set to 1). Densitometry was performed using ImageJ. (D) Western blot analysis was performed on lysates harvested from HFK-31 cells at T 0 or after 24 and 48 h of differentiation in methylcellulose using antibodies to Nbs1 and involucrin. GAPDH was used as a loading control. All results are representative of observations of four or more independent experiments.
    Figure Legend Snippet: Nbs1 is necessary for productive viral replication. (A) DNA was harvested from CIN612 9E cells stably expressing a Scramble shRNA or Nbs1 shRNA at T 0 (undifferentiated) or after 48 and 96 h of differentiation in high-calcium medium. Southern blot analysis was performed to analyze viral genome amplification. The bar graph represents quantification of the episome copy number present at each time point, relative to T 0 shScramble, which was set to 1. Densitometry was performed using ImageJ. Ca, calcium. (B) Total protein was harvested from CIN612 9E shScramble and shNbs1 cells at T 0 or after 48 and 96 h of differentiation in high-calcium medium. Western blot analysis was performed using antibodies to Nbs1 and involucrin, and GAPDH as a loading control. (C) DNA was harvested from human foreskin keratinocytes stably maintaining HPV31 genomes (HFK-31) at T 0 or after 24 and 48 h of differentiation in methylcellulose (MC) and analyzed by Southern blotting for amplification of viral genomes. DNA samples were digested with BamHI (does not cut the viral genome; upper panel) or with HindIII to linearize viral genomes. The bar graph represents quantification of the episome copy number present at each time point, relative to T 0 shScramble (set to 1). Densitometry was performed using ImageJ. (D) Western blot analysis was performed on lysates harvested from HFK-31 cells at T 0 or after 24 and 48 h of differentiation in methylcellulose using antibodies to Nbs1 and involucrin. GAPDH was used as a loading control. All results are representative of observations of four or more independent experiments.

    Techniques Used: Stable Transfection, Expressing, shRNA, Southern Blot, Amplification, Western Blot

    21) Product Images from "Interaction of hemoglobin Grey Lynn (Vientiane) with a non-deletional ?+-thalassemia in an adult Thai proband"

    Article Title: Interaction of hemoglobin Grey Lynn (Vientiane) with a non-deletional ?+-thalassemia in an adult Thai proband

    Journal: Biochemia Medica

    doi: 10.11613/BM.2014.019

    A : Identification of the Hb Grey Lynn mutation by PCR-RFLP assay using Hind III digestion. The locations of primers C1 and B used to generate an α1 specific fragment of 975 bp by PCR are shown. Normal control fragment is digested into two fragments with 627 bp and 348 bp in lengths while the undigested 975 bp fragment indicates the presence of Hb Grey Lynn mutation. M represents the GeneRuler 50 bp Plus DNA ladder. 1: amplified DNA of the proband, 2: Hind III-digested amplified DNA of a normal control, and 3: Hind III-digested amplified DNA of the proband with Hb Grey Lynn. B : Identification of α + -thalassemia [α2 codon 36/37 (−C)] by allele specific PCR. The locations and orientations of primers (αG17 C3) and (αG58 C3) used respectively to produce the 634 bp α2 codon 36/37 (−C) mutation specific fragment and the 391 bp internal control fragment are depicted. M represents the GeneRuler 50 bp Plus DNA ladder. The α2 codon 36/37 (−C) mutation was identified in the proband (lane 2) but not in a normal control (lane 1).
    Figure Legend Snippet: A : Identification of the Hb Grey Lynn mutation by PCR-RFLP assay using Hind III digestion. The locations of primers C1 and B used to generate an α1 specific fragment of 975 bp by PCR are shown. Normal control fragment is digested into two fragments with 627 bp and 348 bp in lengths while the undigested 975 bp fragment indicates the presence of Hb Grey Lynn mutation. M represents the GeneRuler 50 bp Plus DNA ladder. 1: amplified DNA of the proband, 2: Hind III-digested amplified DNA of a normal control, and 3: Hind III-digested amplified DNA of the proband with Hb Grey Lynn. B : Identification of α + -thalassemia [α2 codon 36/37 (−C)] by allele specific PCR. The locations and orientations of primers (αG17 C3) and (αG58 C3) used respectively to produce the 634 bp α2 codon 36/37 (−C) mutation specific fragment and the 391 bp internal control fragment are depicted. M represents the GeneRuler 50 bp Plus DNA ladder. The α2 codon 36/37 (−C) mutation was identified in the proband (lane 2) but not in a normal control (lane 1).

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, RFLP Assay, Amplification

    22) Product Images from "Initiation of Epstein-Barr Virus Lytic Replication Requires Transcription and the Formation of a Stable RNA-DNA Hybrid Molecule at OriLyt ▿"

    Article Title: Initiation of Epstein-Barr Virus Lytic Replication Requires Transcription and the Formation of a Stable RNA-DNA Hybrid Molecule at OriLyt ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02175-10

    RNase H1 impairs OriLyt-dependent plasmid replication. Wild-type pBluescript OriLyt plasmids were cotransfected with BZLF1 into ZKO-293 cells with an RNase H1 expression plasmid or control vector. Plasmids were recovered from cells, digested with (A and B) or without (C and D) HindIII and DpnI enzymes, and analyzed by Southern blotting. Three identical, independent experiments were conducted in each case. One representative experiment is shown for each (A and C). Relative plasmid amounts were quantified using quantitative Southern blotting (B and D). In the cases where enzymes were used, results were calculated as the DpnI-resistant signal over the DpnI-digested signal and were normalized to the value obtained for vector controls (B). In cases where no enzymes were used, results were calculated as replicating (top band) plasmid over supercoiled (bottom band) signal and normalized to the value obtained for vector controls (D). Data averages from all three identical, independent experiments are shown in each case, with error bars representing the standard deviations. Statistical significance was calculated using a two-tailed, unpaired t test. (E) Western blot assays were used to monitor Zta and BALF2 protein expression levels in all transfected cells.
    Figure Legend Snippet: RNase H1 impairs OriLyt-dependent plasmid replication. Wild-type pBluescript OriLyt plasmids were cotransfected with BZLF1 into ZKO-293 cells with an RNase H1 expression plasmid or control vector. Plasmids were recovered from cells, digested with (A and B) or without (C and D) HindIII and DpnI enzymes, and analyzed by Southern blotting. Three identical, independent experiments were conducted in each case. One representative experiment is shown for each (A and C). Relative plasmid amounts were quantified using quantitative Southern blotting (B and D). In the cases where enzymes were used, results were calculated as the DpnI-resistant signal over the DpnI-digested signal and were normalized to the value obtained for vector controls (B). In cases where no enzymes were used, results were calculated as replicating (top band) plasmid over supercoiled (bottom band) signal and normalized to the value obtained for vector controls (D). Data averages from all three identical, independent experiments are shown in each case, with error bars representing the standard deviations. Statistical significance was calculated using a two-tailed, unpaired t test. (E) Western blot assays were used to monitor Zta and BALF2 protein expression levels in all transfected cells.

    Techniques Used: Plasmid Preparation, Expressing, Southern Blot, Two Tailed Test, Western Blot, Transfection

    23) Product Images from "Imprinting at the PLAGL1 domain is contained within a 70-kb CTCF/cohesin-mediated non-allelic chromatin loop"

    Article Title: Imprinting at the PLAGL1 domain is contained within a 70-kb CTCF/cohesin-mediated non-allelic chromatin loop

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks1355

    Chromatin interactions with the CTCF sites upstream of the PLAGL1 -DMR. ( A ) The position of the HindIII sites used for 3C analysis, and annotated ChIA-PET data showing CTCF and POL2 interactions. ( B ) The looping profile in brain (black), placental TCL1 cell line (red) and normal leucocytes (blue) using a constant primer 5 kb upstream of the PLAGL1 -DMR, showing strong interactions with the 3′ UTR and alternative promoters of PLAGL1 . The x-axis shows the position of the primers used. ( C ) The interaction profile in expressing tissues, brain (black) and placental TCL1 cell line (red), using a constant primer between the P3 and P4 promoters. ( D ) Heterozygous SNP rs2064661 in constant fragment 5 kb upstream of the PLAGL1 -DMR allowed for allele-specific chromatin interactions to be assessed in adult brain. Sequence traces reveal biallelic higher-order chromatin interactions between the constant fragment and various contact points throughout the PLAGL1 domain.
    Figure Legend Snippet: Chromatin interactions with the CTCF sites upstream of the PLAGL1 -DMR. ( A ) The position of the HindIII sites used for 3C analysis, and annotated ChIA-PET data showing CTCF and POL2 interactions. ( B ) The looping profile in brain (black), placental TCL1 cell line (red) and normal leucocytes (blue) using a constant primer 5 kb upstream of the PLAGL1 -DMR, showing strong interactions with the 3′ UTR and alternative promoters of PLAGL1 . The x-axis shows the position of the primers used. ( C ) The interaction profile in expressing tissues, brain (black) and placental TCL1 cell line (red), using a constant primer between the P3 and P4 promoters. ( D ) Heterozygous SNP rs2064661 in constant fragment 5 kb upstream of the PLAGL1 -DMR allowed for allele-specific chromatin interactions to be assessed in adult brain. Sequence traces reveal biallelic higher-order chromatin interactions between the constant fragment and various contact points throughout the PLAGL1 domain.

    Techniques Used: ChIA Pet Assay, Expressing, Sequencing

    24) Product Images from "The distribution of DNA translocation times in solid-state nanopores"

    Article Title: The distribution of DNA translocation times in solid-state nanopores

    Journal: Journal of Physics

    doi: 10.1088/0953-8984/22/45/454129

    (A) Event distribution plot of 3 kbp (L C =1020 nm) DNA translocation as a function of solution viscosity in 1.5 M KCl, pH 7.5, and ψ=120 mV. Insert, left axis: the uncertainty in determine DNA chain length due to random walk ΔL/L DNA ( ); right axis: the relative blockade current ΔI/I 0 (◆). The error bars are smaller than the symbols. (B) The drifting speed, (C) the diffusion constant, and (D) the calculated drag force as a function of viscosity.
    Figure Legend Snippet: (A) Event distribution plot of 3 kbp (L C =1020 nm) DNA translocation as a function of solution viscosity in 1.5 M KCl, pH 7.5, and ψ=120 mV. Insert, left axis: the uncertainty in determine DNA chain length due to random walk ΔL/L DNA ( ); right axis: the relative blockade current ΔI/I 0 (◆). The error bars are smaller than the symbols. (B) The drifting speed, (C) the diffusion constant, and (D) the calculated drag force as a function of viscosity.

    Techniques Used: Translocation Assay, Diffusion-based Assay

    (A) Illustration of linear DNA translocation experiment. (B) Typical 3 kbp DNA translocation events in a 8±2 nm diameter pore in 1.5 MKCl with 30% glycerol at pH 7.5. (C) All events distribution plot of current drop ΔI b vs translocation times t d . (D) Selected linear translocation events plot from the data shown in (C).
    Figure Legend Snippet: (A) Illustration of linear DNA translocation experiment. (B) Typical 3 kbp DNA translocation events in a 8±2 nm diameter pore in 1.5 MKCl with 30% glycerol at pH 7.5. (C) All events distribution plot of current drop ΔI b vs translocation times t d . (D) Selected linear translocation events plot from the data shown in (C).

    Techniques Used: Translocation Assay

    (A) Event distribution plot of 3 kbp DNA translocation as a function of applied voltage. Insert, left axis: the uncertainty in determine DNA chain length due to random walk ΔL/L DNA ( ); right axis: the relative blockade current ΔI/I 0 (◆). The error bars are smaller than the symbols. (B) The drifting speed, (C) the diffusion constant, and (D) the calculated drag force as a function of voltage. The experiment was performed in 1.6 M KCl with 20% glycerol at pH 7.5 in a 8±2 nm silicon nitride pore.
    Figure Legend Snippet: (A) Event distribution plot of 3 kbp DNA translocation as a function of applied voltage. Insert, left axis: the uncertainty in determine DNA chain length due to random walk ΔL/L DNA ( ); right axis: the relative blockade current ΔI/I 0 (◆). The error bars are smaller than the symbols. (B) The drifting speed, (C) the diffusion constant, and (D) the calculated drag force as a function of voltage. The experiment was performed in 1.6 M KCl with 20% glycerol at pH 7.5 in a 8±2 nm silicon nitride pore.

    Techniques Used: Translocation Assay, Diffusion-based Assay

    (A) Event distribution plot of translocation of a DNA ladder (λ cut) that contains a mixture of ~2.17, 4.36, 6.56, 9.42, and 23 kbp DNA. The experiment was performed in 1.0 M KCl with no glycerol. More than 20,000 events are in this event distribution plot. (B) The experiment was performed in 1.6 M KCl with 20% glycerol. Both sets of data were recorded with low pass filter set at 100 kHz. The fitted drifting speed (C) and the diffusion constants (D) as a function of the DNA chain length. All data are measured with 10±2 nm silicon nitride pores and the applied voltage was 120 mV. The error bars for drifting speed in panel C are smaller than the markers for all the data points.
    Figure Legend Snippet: (A) Event distribution plot of translocation of a DNA ladder (λ cut) that contains a mixture of ~2.17, 4.36, 6.56, 9.42, and 23 kbp DNA. The experiment was performed in 1.0 M KCl with no glycerol. More than 20,000 events are in this event distribution plot. (B) The experiment was performed in 1.6 M KCl with 20% glycerol. Both sets of data were recorded with low pass filter set at 100 kHz. The fitted drifting speed (C) and the diffusion constants (D) as a function of the DNA chain length. All data are measured with 10±2 nm silicon nitride pores and the applied voltage was 120 mV. The error bars for drifting speed in panel C are smaller than the markers for all the data points.

    Techniques Used: Translocation Assay, Diffusion-based Assay

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

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

    Journal: The Journal of Experimental Medicine

    doi:

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

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

    26) Product Images from "Tumor treating fields (TTFields) delay DNA damage repair following radiation treatment of glioma cells"

    Article Title: Tumor treating fields (TTFields) delay DNA damage repair following radiation treatment of glioma cells

    Journal: Radiation Oncology (London, England)

    doi: 10.1186/s13014-017-0941-6

    TTFields Influence DNA Damage Repair by Homologous Recombination in Glioma Cells. a pDNA-PKcs (pS2056) and total DNA-PK were compared between U-118 MG cells either untreated or treated with RT or TTFields alone or their combination at indicated time points post RT (4 Gy). Lamin B was used as loading control. b U-118 MG cells were transfected with an intact pGL2-Luc vector or vector that was linearized with either HindIII or EcoRI. Luc activity was measured in cells prior and post 24 h TTFields treatment. c - d U-118 MG cells were irradiated with 4 Gy and immediately treated with TTFields for 1 h, 2 h, or 24 h. c Rad 51 foci formation was analyzed by immunofluorescence at 24 h post treatment. Rad 51 foci (Red) and DAPI (blue) stained nuclei are shown. Scale bar - 5 μm. d The average Rad51 foci in cells with more than 5 foci are shown
    Figure Legend Snippet: TTFields Influence DNA Damage Repair by Homologous Recombination in Glioma Cells. a pDNA-PKcs (pS2056) and total DNA-PK were compared between U-118 MG cells either untreated or treated with RT or TTFields alone or their combination at indicated time points post RT (4 Gy). Lamin B was used as loading control. b U-118 MG cells were transfected with an intact pGL2-Luc vector or vector that was linearized with either HindIII or EcoRI. Luc activity was measured in cells prior and post 24 h TTFields treatment. c - d U-118 MG cells were irradiated with 4 Gy and immediately treated with TTFields for 1 h, 2 h, or 24 h. c Rad 51 foci formation was analyzed by immunofluorescence at 24 h post treatment. Rad 51 foci (Red) and DAPI (blue) stained nuclei are shown. Scale bar - 5 μm. d The average Rad51 foci in cells with more than 5 foci are shown

    Techniques Used: Homologous Recombination, Transfection, Plasmid Preparation, Activity Assay, Irradiation, Immunofluorescence, Staining

    27) Product Images from "5C analysis of the Epidermal Differentiation Complex locus reveals distinct chromatin interaction networks between gene-rich and gene-poor TADs in skin epithelial cells"

    Article Title: 5C analysis of the Epidermal Differentiation Complex locus reveals distinct chromatin interaction networks between gene-rich and gene-poor TADs in skin epithelial cells

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006966

    Gene-rich and gene-poor regions are organized into distinct TADs at the EDC locus in keratinocytes. (a) Schematic structure of the 5.3 Mb genomic region containing the EDC locus on mouse chromosome 3 analysed using 5C technology in this manuscript (mm9/chr.3:89,900,000–95,200,000). (b) Alternating 5C probe design for the unique HindIII sites in the interrogated genomic regions. The position of the restriction sites interrogated by the forward primers are shown in blue, interrogated by the reverse primers are shown in red and the site for which the primers could not be designed are shown in green. (c) Heatmaps representing raw 5C data for both KC replicates. Reverse probes are plotted as columns and the forward probes as rows. Pearson’s correlation coefficient is also shown. (d) Heatmap representing the 5C data after the normalization and binning (bin size 150 kb, step size 15kb) in KCs. The position of TAD border midpoints (average for the midpoints calculated based on the insulation index analysis in two replicates independently) are identified by green lines under the heatmaps. Note the high frequency of the spatial contacts between the gene-poor TADs 2 and 5 (indicated by dashed rectangle on the heat map). The position of the regions covered by the BAC fish probes used in these studies, schematic map of the studied locus and insulation indexes profiles for two 5C library replicates are also shown. (e) Multi-colour 3D FISH analysis with BAC probes A (located at the 5’ border of TAD3, B (located at the 3’ border of TAD4) and C (located within TAD4) (left) , or with BAC probe D (located within gene-poor TAD2) and E (located within gene-poor TAD5) (right) in basal epidermal keratinocytes. Representative single optical sections are shown. Scale bars are 2μm. (f) Box plots showing median, 25% quartile, 75% quartile with whiskers indicating maximum and minimum for spatial distances between the centres of the regions covered by probes A and B, probes B and C, as well as probes D and E before (in μm) and after normalization to the average nuclear radius (in % of average nuclear radius) in basal epidermal keratinocytes in situ . The distances between the centres of the regions covered by the probes A and B (located within TAD3) are significantly shorter than the distances between loci covered by the probes B and C (located within TAD4). The indicated p-values for pair-wise comparison are calculated using Mann-Whitney U-test, n = 60 alleles for each interrogated locus. The distances between the centres of the regions covered by the probes D and E (located in the gene poor TADs 2 and 5 respectively) are similar to the much closer regions covered by the probes B and C (located in the adjacent gene-rich TAD3 and TAD4).
    Figure Legend Snippet: Gene-rich and gene-poor regions are organized into distinct TADs at the EDC locus in keratinocytes. (a) Schematic structure of the 5.3 Mb genomic region containing the EDC locus on mouse chromosome 3 analysed using 5C technology in this manuscript (mm9/chr.3:89,900,000–95,200,000). (b) Alternating 5C probe design for the unique HindIII sites in the interrogated genomic regions. The position of the restriction sites interrogated by the forward primers are shown in blue, interrogated by the reverse primers are shown in red and the site for which the primers could not be designed are shown in green. (c) Heatmaps representing raw 5C data for both KC replicates. Reverse probes are plotted as columns and the forward probes as rows. Pearson’s correlation coefficient is also shown. (d) Heatmap representing the 5C data after the normalization and binning (bin size 150 kb, step size 15kb) in KCs. The position of TAD border midpoints (average for the midpoints calculated based on the insulation index analysis in two replicates independently) are identified by green lines under the heatmaps. Note the high frequency of the spatial contacts between the gene-poor TADs 2 and 5 (indicated by dashed rectangle on the heat map). The position of the regions covered by the BAC fish probes used in these studies, schematic map of the studied locus and insulation indexes profiles for two 5C library replicates are also shown. (e) Multi-colour 3D FISH analysis with BAC probes A (located at the 5’ border of TAD3, B (located at the 3’ border of TAD4) and C (located within TAD4) (left) , or with BAC probe D (located within gene-poor TAD2) and E (located within gene-poor TAD5) (right) in basal epidermal keratinocytes. Representative single optical sections are shown. Scale bars are 2μm. (f) Box plots showing median, 25% quartile, 75% quartile with whiskers indicating maximum and minimum for spatial distances between the centres of the regions covered by probes A and B, probes B and C, as well as probes D and E before (in μm) and after normalization to the average nuclear radius (in % of average nuclear radius) in basal epidermal keratinocytes in situ . The distances between the centres of the regions covered by the probes A and B (located within TAD3) are significantly shorter than the distances between loci covered by the probes B and C (located within TAD4). The indicated p-values for pair-wise comparison are calculated using Mann-Whitney U-test, n = 60 alleles for each interrogated locus. The distances between the centres of the regions covered by the probes D and E (located in the gene poor TADs 2 and 5 respectively) are similar to the much closer regions covered by the probes B and C (located in the adjacent gene-rich TAD3 and TAD4).

    Techniques Used: BAC Assay, Fluorescence In Situ Hybridization, In Situ, MANN-WHITNEY

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv1345

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

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

    29) Product Images from "Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants"

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190526

    Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with Bam HI and Hin dIII to obtain pCAMBIA_ASX. (B) T35S–AttR2– ccd B–AttR1 cassette was released from the Gateway plasmid backbone by digestion with Xba I and Sac I and replaced with a I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Spec: selection marker conferring spectinomycin resistance in E . coli and A . tumefaciens , Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, Hin dIII, I- Sce I, Bam HI, Xba I, Sac I: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method.
    Figure Legend Snippet: Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with Bam HI and Hin dIII to obtain pCAMBIA_ASX. (B) T35S–AttR2– ccd B–AttR1 cassette was released from the Gateway plasmid backbone by digestion with Xba I and Sac I and replaced with a I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Spec: selection marker conferring spectinomycin resistance in E . coli and A . tumefaciens , Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, Hin dIII, I- Sce I, Bam HI, Xba I, Sac I: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method.

    Techniques Used: Expressing, Plasmid Preparation, Clone Assay, Selection, Marker, Ligation

    30) Product Images from "Rescue of Infectious Recombinant Hazara Nairovirus from cDNA Reveals the Nucleocapsid Protein DQVD Caspase Cleavage Motif Performs an Essential Role other than Cleavage"

    Article Title: Rescue of Infectious Recombinant Hazara Nairovirus from cDNA Reveals the Nucleocapsid Protein DQVD Caspase Cleavage Motif Performs an Essential Role other than Cleavage

    Journal: Journal of Virology

    doi: 10.1128/JVI.00616-19

    Confirmation of cDNA origin via recovery of mutant rHAZV. (A) Table outlining the change (underlined) to the cDNA sequence of the HAZV-N ORF and the resulting amino acid sequence. (B) Detection of HAZV-N by Western blotting posttransfection (p.Tr) of BSR-T7 cells and 48 h postinfection (p.Inf). Supernatant samples collected from transfected BSR-T7 cells at 96 h posttransfection were used to infect monolayers of SW13 cells. Following a 48-h infection, lysates were collected and analyzed by Western blotting for N expression. Recovery of rHAZV containing a HindIII restriction site (rHAZV-G723T) was carried out alongside complete control recovery of rHAZV. Detection of GAPDH abundance was included as a loading control. (C) Schematic showing the location of the inserted HindIII restriction site in the S segment ORF cDNA. UTR, untranslated region. (D) Restriction digest of double-stranded DNA fragments following RNA extraction of rHAZV- and rHAZV-G723T-containing supernatants, first-strand synthesis, and PCR amplification of viral genetic material.
    Figure Legend Snippet: Confirmation of cDNA origin via recovery of mutant rHAZV. (A) Table outlining the change (underlined) to the cDNA sequence of the HAZV-N ORF and the resulting amino acid sequence. (B) Detection of HAZV-N by Western blotting posttransfection (p.Tr) of BSR-T7 cells and 48 h postinfection (p.Inf). Supernatant samples collected from transfected BSR-T7 cells at 96 h posttransfection were used to infect monolayers of SW13 cells. Following a 48-h infection, lysates were collected and analyzed by Western blotting for N expression. Recovery of rHAZV containing a HindIII restriction site (rHAZV-G723T) was carried out alongside complete control recovery of rHAZV. Detection of GAPDH abundance was included as a loading control. (C) Schematic showing the location of the inserted HindIII restriction site in the S segment ORF cDNA. UTR, untranslated region. (D) Restriction digest of double-stranded DNA fragments following RNA extraction of rHAZV- and rHAZV-G723T-containing supernatants, first-strand synthesis, and PCR amplification of viral genetic material.

    Techniques Used: Mutagenesis, Sequencing, Western Blot, Transfection, Infection, Expressing, RNA Extraction, Polymerase Chain Reaction, Amplification

    31) Product Images from "Genetic Environments of the rmtA Gene in Pseudomonas aeruginosa Clinical Isolates"

    Article Title: Genetic Environments of the rmtA Gene in Pseudomonas aeruginosa Clinical Isolates

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.48.6.2069-2074.2004

    Comparison of the genetic organizations of AR-2 and AR-11. Double-headed striped arrows indicate the position of the rmtA locus and that of the region common to both sequenced areas. Inserts of pBCRMTH2, pBCRMTE2, and pBCRMTE11 are indicated by horizontal lines. Rectangles filled with wavy lines, sequences similar to part of Tn 5041 . Solid arrowheads in the 15.8-kbp EcoRI fragment, terminal inverted repeats. mer , the mercury resistance operon, includes merR . Sequence 1, transposase gene-like sequence; sequence 2, Na + /H + antiporter-like sequence; orfA , probable tRNA ribosyltransferase gene; orfQ ′, part of orfQ ; orfA ′, part of orfA ; IR, probable inverted repeat. Restriction sites: H, HindIII; E, EcoRI. Sequences 1 and 2 encode no complete proteins due to several frameshifts and deletions.
    Figure Legend Snippet: Comparison of the genetic organizations of AR-2 and AR-11. Double-headed striped arrows indicate the position of the rmtA locus and that of the region common to both sequenced areas. Inserts of pBCRMTH2, pBCRMTE2, and pBCRMTE11 are indicated by horizontal lines. Rectangles filled with wavy lines, sequences similar to part of Tn 5041 . Solid arrowheads in the 15.8-kbp EcoRI fragment, terminal inverted repeats. mer , the mercury resistance operon, includes merR . Sequence 1, transposase gene-like sequence; sequence 2, Na + /H + antiporter-like sequence; orfA , probable tRNA ribosyltransferase gene; orfQ ′, part of orfQ ; orfA ′, part of orfA ; IR, probable inverted repeat. Restriction sites: H, HindIII; E, EcoRI. Sequences 1 and 2 encode no complete proteins due to several frameshifts and deletions.

    Techniques Used: Sequencing

    32) Product Images from "Transcription-Dependent Gene Looping of the HIV-1 Provirus Is Dictated by Recognition of Pre-mRNA Processing Signals"

    Article Title: Transcription-Dependent Gene Looping of the HIV-1 Provirus Is Dictated by Recognition of Pre-mRNA Processing Signals

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2007.11.030

    Integrated U1 HIV-1 Proviruses Form Quantitatively Different Looping Conformations (A) Representation of integrated provirus and flanking chromosomal sequence with restriction enzyme sites and primers for BanI and HindIII 3C analysis. Numbers denote distance from 5′ (−) or 3′ (+) proviral ends. Arrows indicate primer direction and name; black/gray arrows refer to primers that detect LTR and the MSD, respectively. HIV-1 long-terminal repeat (LTR) regions (U3, R, and U5), MSD, and polyadenylation sites (pA) are indicated. (B) int-ChrX 3C. Unstimulated (−TPA), cells after 5 hr TPA (+TPA), and control PCR panel (control). Positive lanes (+) signify internal HIV-1 PCR controls on U1 gDNA (control panel) and chromatin (for −/+TPA; see Experimental Procedures ). Common PCR primers are shown above the figure, with the second primer shown above each lane. Graphs below represent quantified percentages of 3C product observed compared to PCR control, standardized between − and +TPA samples (using internal PCR controls; see + lane). (C) Quantitative analysis of Tat- or TPA-induced int-ChrX and int-Chr2 loop structures. (a) HIV-1 qRT-PCR at 0, 3, and 5 hr post-TPA treatment with nuc1 primers ( Figure 1 A) standardized to 18S rRNA transcription. (b) q3C HindIII-digested U1 chromatin analysis; primers used to detect the “loop”: interaction (primers 22/23 for int-Chr2 and X2/X3 for int-ChrX) compared to the adjacent amplified fragment (primers 22/H7 or X2/H7). (D) (a) HIV-1 qRT-PCR treated with 0 (10 μg GFP control), 5, or 10 μg of GFP-tagged Tat protein. (b) q3C HindIII analysis of U1 chromatin treated with 10 μg Tat-GFP or GFP. Error bars represent SEM from n = 6 samples from two separate chromatin preparations (except 22/23 and X2/X3 in Tat induction analysis where n = 9, from three separate chromatin preparations).
    Figure Legend Snippet: Integrated U1 HIV-1 Proviruses Form Quantitatively Different Looping Conformations (A) Representation of integrated provirus and flanking chromosomal sequence with restriction enzyme sites and primers for BanI and HindIII 3C analysis. Numbers denote distance from 5′ (−) or 3′ (+) proviral ends. Arrows indicate primer direction and name; black/gray arrows refer to primers that detect LTR and the MSD, respectively. HIV-1 long-terminal repeat (LTR) regions (U3, R, and U5), MSD, and polyadenylation sites (pA) are indicated. (B) int-ChrX 3C. Unstimulated (−TPA), cells after 5 hr TPA (+TPA), and control PCR panel (control). Positive lanes (+) signify internal HIV-1 PCR controls on U1 gDNA (control panel) and chromatin (for −/+TPA; see Experimental Procedures ). Common PCR primers are shown above the figure, with the second primer shown above each lane. Graphs below represent quantified percentages of 3C product observed compared to PCR control, standardized between − and +TPA samples (using internal PCR controls; see + lane). (C) Quantitative analysis of Tat- or TPA-induced int-ChrX and int-Chr2 loop structures. (a) HIV-1 qRT-PCR at 0, 3, and 5 hr post-TPA treatment with nuc1 primers ( Figure 1 A) standardized to 18S rRNA transcription. (b) q3C HindIII-digested U1 chromatin analysis; primers used to detect the “loop”: interaction (primers 22/23 for int-Chr2 and X2/X3 for int-ChrX) compared to the adjacent amplified fragment (primers 22/H7 or X2/H7). (D) (a) HIV-1 qRT-PCR treated with 0 (10 μg GFP control), 5, or 10 μg of GFP-tagged Tat protein. (b) q3C HindIII analysis of U1 chromatin treated with 10 μg Tat-GFP or GFP. Error bars represent SEM from n = 6 samples from two separate chromatin preparations (except 22/23 and X2/X3 in Tat induction analysis where n = 9, from three separate chromatin preparations).

    Techniques Used: Sequencing, Polymerase Chain Reaction, Quantitative RT-PCR, Amplification

    33) Product Images from "Modulation of RNase E Activity by Alternative RNA Binding Sites"

    Article Title: Modulation of RNase E Activity by Alternative RNA Binding Sites

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0090610

    Effects of Y25A and Q36R on the catalytic activity of RNase E in vivo and in vitro . (A) Plasmid copy number of pNRNE4, pNRNE4-Q36R and pNRNE4-Y25A in KSL2000. Plasmids were purified from KSL2000 cells harboring pNRNE4, pNRNE4-Q36R or pNRNE4-Y25A and were digested with Hin dIII, which has a unique cleavage site in all of the plasmids tested. Plasmid copy number was calculated relative to the concurrent presence of the pSC101 derivative (pBAD-RNE), which replicates independently of Rne, by measuring the molar ratio of the ColE1-type plasmid to the pBAD-RNE plasmid. (B) Growth characteristics of KSL2003 cells expressing wild-type N-Rne or the Q36R or Y25A mutant proteins. Growth of KSL2003 cells harboring pLAC-RNE2, pLAC-RNE2-Q36R, or pLAC-RNE2-Y25A was measured individually on LB-agar plates containing 1.0 to 1000 µM IPTG. Numbers on the top indicate the number of bacterial cells in each spot. (C) Plasmid copy number of pET28a in KSL2003. Plasmids were purified from KSL2003, KSL2003-Q36R or KSL2003-Y25A cells harboring pET28a and digested with Hin dIII, which has a unique cleavage site in all the plasmids tested. Plasmid copy number was calculated relative to the concurrent presence of the pSC101 derivative (pLAC-RNE2, pLAC-RNE2-Q36R or pLAC-RNE2-Y25A) by measuring the molar ratio of the ColE1-type plasmid to the pSC101-derived plasmid. (D) Expression profiles of Rne and mutant proteins in KSL2003. The membrane probed with an anti-Rne polyclonal antibody was stripped and reprobed with an anti-S1 polyclonal antibody to provide an internal standard. The relative abundance of protein was quantified by setting the amount of wild-type Rne to 1. KSL2003 cells were grown in LB medium containing 10 µM IPTG. (E) In vitro cleavage of p-BR13 by wild-type N-Rne, Q36R and Y25A mutant proteins. Two pmol of 5′ end-labeled p-BR13 was incubated with 1 pmol of purified wild-type N-Rne or Q36R or Y25A mutant protein in 20 µl of cleavage buffer at 37°C. Samples were removed at each indicated time point and mixed with an equal volume of loading buffer. Samples were denatured at 65°C for 5 min and loaded onto 15% polyacrylamide gel containing 8 M urea. The radioactivity in each band was quantified using a phosphorimager and OptiQuant software.
    Figure Legend Snippet: Effects of Y25A and Q36R on the catalytic activity of RNase E in vivo and in vitro . (A) Plasmid copy number of pNRNE4, pNRNE4-Q36R and pNRNE4-Y25A in KSL2000. Plasmids were purified from KSL2000 cells harboring pNRNE4, pNRNE4-Q36R or pNRNE4-Y25A and were digested with Hin dIII, which has a unique cleavage site in all of the plasmids tested. Plasmid copy number was calculated relative to the concurrent presence of the pSC101 derivative (pBAD-RNE), which replicates independently of Rne, by measuring the molar ratio of the ColE1-type plasmid to the pBAD-RNE plasmid. (B) Growth characteristics of KSL2003 cells expressing wild-type N-Rne or the Q36R or Y25A mutant proteins. Growth of KSL2003 cells harboring pLAC-RNE2, pLAC-RNE2-Q36R, or pLAC-RNE2-Y25A was measured individually on LB-agar plates containing 1.0 to 1000 µM IPTG. Numbers on the top indicate the number of bacterial cells in each spot. (C) Plasmid copy number of pET28a in KSL2003. Plasmids were purified from KSL2003, KSL2003-Q36R or KSL2003-Y25A cells harboring pET28a and digested with Hin dIII, which has a unique cleavage site in all the plasmids tested. Plasmid copy number was calculated relative to the concurrent presence of the pSC101 derivative (pLAC-RNE2, pLAC-RNE2-Q36R or pLAC-RNE2-Y25A) by measuring the molar ratio of the ColE1-type plasmid to the pSC101-derived plasmid. (D) Expression profiles of Rne and mutant proteins in KSL2003. The membrane probed with an anti-Rne polyclonal antibody was stripped and reprobed with an anti-S1 polyclonal antibody to provide an internal standard. The relative abundance of protein was quantified by setting the amount of wild-type Rne to 1. KSL2003 cells were grown in LB medium containing 10 µM IPTG. (E) In vitro cleavage of p-BR13 by wild-type N-Rne, Q36R and Y25A mutant proteins. Two pmol of 5′ end-labeled p-BR13 was incubated with 1 pmol of purified wild-type N-Rne or Q36R or Y25A mutant protein in 20 µl of cleavage buffer at 37°C. Samples were removed at each indicated time point and mixed with an equal volume of loading buffer. Samples were denatured at 65°C for 5 min and loaded onto 15% polyacrylamide gel containing 8 M urea. The radioactivity in each band was quantified using a phosphorimager and OptiQuant software.

    Techniques Used: Activity Assay, In Vivo, In Vitro, Plasmid Preparation, Purification, Expressing, Mutagenesis, Derivative Assay, Labeling, Incubation, Radioactivity, Software

    34) Product Images from "A view through a chromatin loop: insights into the ecdysone activation of early genes in Drosophila"

    Article Title: A view through a chromatin loop: insights into the ecdysone activation of early genes in Drosophila

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku754

    Long-distance interactions at the E75 gene in imaginal discs. 3C analysis at the E75 locus was performed in imaginal discs using HindIII. Schematic of the E75 locus is shown at the top. Horizontal arrows indicate TSSs and vertical bars indicate HindIII sites. EcR binding peaks (A–L) from whole larvae at 0 APF, derived from the modENCODE database, are denoted by red boxes at the top of the diagram. Cross-linking frequencies ( y- axis) between the fixed HindIII anchor for E75B (upper panel), E75A (middle panel) or E75C (lower panel) and the rest of the locus were measured in late third instar imaginal discs. Location of fixed anchor sites for E75B , E75A and E75C are marked by red, blue and green bars, respectively, and test sites are marked by gray bars. Black angle brackets (‘
    Figure Legend Snippet: Long-distance interactions at the E75 gene in imaginal discs. 3C analysis at the E75 locus was performed in imaginal discs using HindIII. Schematic of the E75 locus is shown at the top. Horizontal arrows indicate TSSs and vertical bars indicate HindIII sites. EcR binding peaks (A–L) from whole larvae at 0 APF, derived from the modENCODE database, are denoted by red boxes at the top of the diagram. Cross-linking frequencies ( y- axis) between the fixed HindIII anchor for E75B (upper panel), E75A (middle panel) or E75C (lower panel) and the rest of the locus were measured in late third instar imaginal discs. Location of fixed anchor sites for E75B , E75A and E75C are marked by red, blue and green bars, respectively, and test sites are marked by gray bars. Black angle brackets (‘

    Techniques Used: Binding Assay, Derivative Assay

    Long-distance 3C interactions at the E75 gene. Schematic of the E75 locus is shown at the top. Horizontal arrows indicate TSSs, vertical bars indicate HindIII sites and numbered arrows designate EcREs. Cross-linking frequencies ( y- axis) between the fixed HindIII anchor for E75B (upper panel), E75A (middle panel), or E75C (lower panel) and the rest of the locus were measured in S2 cells in the absence or presence of ecdysone as indicated. Location of fixed anchor sites for E75B , E75A and E75C are marked by red, blue and green bars, respectively, and test sites are marked by gray bars. Black angle brackets (‘
    Figure Legend Snippet: Long-distance 3C interactions at the E75 gene. Schematic of the E75 locus is shown at the top. Horizontal arrows indicate TSSs, vertical bars indicate HindIII sites and numbered arrows designate EcREs. Cross-linking frequencies ( y- axis) between the fixed HindIII anchor for E75B (upper panel), E75A (middle panel), or E75C (lower panel) and the rest of the locus were measured in S2 cells in the absence or presence of ecdysone as indicated. Location of fixed anchor sites for E75B , E75A and E75C are marked by red, blue and green bars, respectively, and test sites are marked by gray bars. Black angle brackets (‘

    Techniques Used:

    Long-distance 3C interactions at the Broad locus. Schematic of Broad is shown at the top. Horizontal arrow indicates the TSS, vertical bars indicate HindIII sites and numbered arrows designate EcREs. Cross-linking frequencies ( y -axis) between the fixed HindIII anchor at the proximal promoter and the rest of the locus were measured in S2 cells in the absence or presence of ecdysone as indicated. Locations of the fixed anchor site and test sites are marked by blue and gray bars, respectively. Black angle bracket (‘ > ’) indicates the location and direction of the anchor primer. Blue brackets indicate the location and direction of test primers in fragments that interact with the Broad promoter, either in the presence or absence of ecdysone. Coordinates are given along the x -axis relative to the proximal TSS. Asterisk indicates significant difference between ecdysone and control samples (Paired Student's t -test, P
    Figure Legend Snippet: Long-distance 3C interactions at the Broad locus. Schematic of Broad is shown at the top. Horizontal arrow indicates the TSS, vertical bars indicate HindIII sites and numbered arrows designate EcREs. Cross-linking frequencies ( y -axis) between the fixed HindIII anchor at the proximal promoter and the rest of the locus were measured in S2 cells in the absence or presence of ecdysone as indicated. Locations of the fixed anchor site and test sites are marked by blue and gray bars, respectively. Black angle bracket (‘ > ’) indicates the location and direction of the anchor primer. Blue brackets indicate the location and direction of test primers in fragments that interact with the Broad promoter, either in the presence or absence of ecdysone. Coordinates are given along the x -axis relative to the proximal TSS. Asterisk indicates significant difference between ecdysone and control samples (Paired Student's t -test, P

    Techniques Used:

    Effect of EcR knockdown on pre-existing promoter–EcRE interactions at the E75 and Broad loci. S2 cells were incubated for 3 days with or without dsRNA targeting EcR , then incubated with or without 1×10 −6 M ecdysone as indicated. Each sample was divided and used in qRT-PCR and 3C analyses. HindIII-based 3C analysis was performed, and interactions were measured between the E75 (A) or Broad (B) promoters and the EcRE(s) indicated ( x- axis). Cross-linking frequencies ( y- axis) are shown relative to the control sample. Bars indicate mean ± SEM from three independent experiments. Inset: total RNA was extracted and transcript abundance was measured by qRT-PCR for E75B , E75A , E75C and Broad core. Expression was normalized to the rp49 transcript and is shown relative to the control sample ( y -axis). Bars indicate mean ± SEM from three independent experiments. Number above each bar indicates fold change relative to control sample.
    Figure Legend Snippet: Effect of EcR knockdown on pre-existing promoter–EcRE interactions at the E75 and Broad loci. S2 cells were incubated for 3 days with or without dsRNA targeting EcR , then incubated with or without 1×10 −6 M ecdysone as indicated. Each sample was divided and used in qRT-PCR and 3C analyses. HindIII-based 3C analysis was performed, and interactions were measured between the E75 (A) or Broad (B) promoters and the EcRE(s) indicated ( x- axis). Cross-linking frequencies ( y- axis) are shown relative to the control sample. Bars indicate mean ± SEM from three independent experiments. Inset: total RNA was extracted and transcript abundance was measured by qRT-PCR for E75B , E75A , E75C and Broad core. Expression was normalized to the rp49 transcript and is shown relative to the control sample ( y -axis). Bars indicate mean ± SEM from three independent experiments. Number above each bar indicates fold change relative to control sample.

    Techniques Used: Incubation, Quantitative RT-PCR, Expressing

    Long-distance interactions at the E75 gene in fat body. 3C analysis at the E75 locus was performed in the fat body using HindIII. Schematic of the E75 locus is shown at the top. Horizontal arrows indicate TSSs and vertical bars indicate HindIII sites. EcR binding peaks (A–L) from whole larvae at 0 APF, derived from the modENCODE database, are denoted by red boxes at the top of the diagram. Cross-linking frequencies ( y- axis) between the fixed HindIII anchor for E75B (upper panel), E75A (middle panel) or E75C (lower panel) and the rest of the locus were measured in late third instar or mid-third instar fat bodies as indicated. Location of fixed anchor sites for E75B , E75A and E75C are marked by red, blue and green bars, respectively, and test sites are marked by gray bars. Black angle brackets (‘
    Figure Legend Snippet: Long-distance interactions at the E75 gene in fat body. 3C analysis at the E75 locus was performed in the fat body using HindIII. Schematic of the E75 locus is shown at the top. Horizontal arrows indicate TSSs and vertical bars indicate HindIII sites. EcR binding peaks (A–L) from whole larvae at 0 APF, derived from the modENCODE database, are denoted by red boxes at the top of the diagram. Cross-linking frequencies ( y- axis) between the fixed HindIII anchor for E75B (upper panel), E75A (middle panel) or E75C (lower panel) and the rest of the locus were measured in late third instar or mid-third instar fat bodies as indicated. Location of fixed anchor sites for E75B , E75A and E75C are marked by red, blue and green bars, respectively, and test sites are marked by gray bars. Black angle brackets (‘

    Techniques Used: Binding Assay, Derivative Assay

    Long-distance interactions at the Broad locus in larval tissues. Schematic of Broad is shown at the top. Horizontal arrow indicates TSS and vertical bars indicate HindIII sites. EcR binding peaks (A–E) from whole larvae at 0 APF, derived from the modENCODE database, are denoted by red boxes at the top of the diagram. Cross-linking frequencies ( y- axis) between the fixed HindIII anchor for Broad and the rest of the locus were measured in late third instar imaginal discs (upper panel) and in late and mid-third instar fat bodies (lower panel). Location of the fixed anchor site and test sites are marked by blue and gray bars, respectively. Black angle brackets (‘ > ’) indicate the location and direction of anchor primers. Blue brackets indicate the location and direction of test primers in fragments that interact with the Broad promoter. Coordinates are given along the x- axis relative to the proximal TSS. Asterisk indicates significant difference between late and mid-third instar fat bodies (Unpaired Student's t -test, P
    Figure Legend Snippet: Long-distance interactions at the Broad locus in larval tissues. Schematic of Broad is shown at the top. Horizontal arrow indicates TSS and vertical bars indicate HindIII sites. EcR binding peaks (A–E) from whole larvae at 0 APF, derived from the modENCODE database, are denoted by red boxes at the top of the diagram. Cross-linking frequencies ( y- axis) between the fixed HindIII anchor for Broad and the rest of the locus were measured in late third instar imaginal discs (upper panel) and in late and mid-third instar fat bodies (lower panel). Location of the fixed anchor site and test sites are marked by blue and gray bars, respectively. Black angle brackets (‘ > ’) indicate the location and direction of anchor primers. Blue brackets indicate the location and direction of test primers in fragments that interact with the Broad promoter. Coordinates are given along the x- axis relative to the proximal TSS. Asterisk indicates significant difference between late and mid-third instar fat bodies (Unpaired Student's t -test, P

    Techniques Used: Binding Assay, Derivative Assay

    35) Product Images from "Mechanism of Start Site Selection by RNA Polymerase II"

    Article Title: Mechanism of Start Site Selection by RNA Polymerase II

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.281576

    Effects of ssl2-508 on gene looping. A , schematic depiction of SEN1 , BLM10 , and HEM3 genes, including the positions of the HindIII sites and the P1 and T1 primer pairs. Approximate length of each ORF is indicated as kb. For description of the 3C assay,
    Figure Legend Snippet: Effects of ssl2-508 on gene looping. A , schematic depiction of SEN1 , BLM10 , and HEM3 genes, including the positions of the HindIII sites and the P1 and T1 primer pairs. Approximate length of each ORF is indicated as kb. For description of the 3C assay,

    Techniques Used:

    36) Product Images from "Cloning and Characterization of an Armillaria gallica cDNA Encoding Protoilludene Synthase, Which Catalyzes the First Committed Step in the Synthesis of Antimicrobial Melleolides *"

    Article Title: Cloning and Characterization of an Armillaria gallica cDNA Encoding Protoilludene Synthase, Which Catalyzes the First Committed Step in the Synthesis of Antimicrobial Melleolides *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.165845

    Determination of A. gallica protoilludene synthase gene copy number by Southern blot hybridization. One clear band is visible in the BamHI and EcoRI lanes (neither enzyme has a target site in the genomic clone), whereas two bands are visible in the HindIII lane (which has two target sites in the clone, 85 bp apart).
    Figure Legend Snippet: Determination of A. gallica protoilludene synthase gene copy number by Southern blot hybridization. One clear band is visible in the BamHI and EcoRI lanes (neither enzyme has a target site in the genomic clone), whereas two bands are visible in the HindIII lane (which has two target sites in the clone, 85 bp apart).

    Techniques Used: Southern Blot, Hybridization

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

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

    Journal: mBio

    doi: 10.1128/mBio.01298-18

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

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

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

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

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.186.10.3015-3021.2004

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

    Techniques Used: Footprinting, Binding Assay, Labeling, Incubation

    39) Product Images from "DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats"

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats

    Journal: bioRxiv

    doi: 10.1101/2020.06.20.162743

    Pooled measurement of DNA polymerase stalling at STRs. (a) Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structures annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. (b) Extended and stalled products were then analysed by denaturing Poly Acrylamide Gel (PAGE) electrophoresis, recovered from the gel matrix and prepared for high throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence form the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, are reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear overtime.
    Figure Legend Snippet: Pooled measurement of DNA polymerase stalling at STRs. (a) Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structures annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. (b) Extended and stalled products were then analysed by denaturing Poly Acrylamide Gel (PAGE) electrophoresis, recovered from the gel matrix and prepared for high throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence form the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, are reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear overtime.

    Techniques Used: High Throughput Screening Assay, Primer Extension Assay, DNA Synthesis, Microarray, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Produced, Acrylamide Gel Assay, Polyacrylamide Gel Electrophoresis, Electrophoresis, Next-Generation Sequencing, Sequencing, Fluorescence, Imaging

    40) Product Images from "Identification of Genes Essential for Prey-Independent Growth of Bdellovibrio bacteriovorus HD100 ▿ HD100 ▿ §"

    Article Title: Identification of Genes Essential for Prey-Independent Growth of Bdellovibrio bacteriovorus HD100 ▿ HD100 ▿ §

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01343-10

    BamHI-HindIII fragment containing a Cm r cassette integrated into the chromosome of B. bacteriovorus HD100 used for construction of knockout plasmid pNR16. The fragment was obtained by PCR amplification using primers Bd29 and Bd30 from chromosomal DNA
    Figure Legend Snippet: BamHI-HindIII fragment containing a Cm r cassette integrated into the chromosome of B. bacteriovorus HD100 used for construction of knockout plasmid pNR16. The fragment was obtained by PCR amplification using primers Bd29 and Bd30 from chromosomal DNA

    Techniques Used: Knock-Out, Plasmid Preparation, Polymerase Chain Reaction, Amplification

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Liver fluke infections by Amphimerus sp. (Digenea: Opisthorchiidae) in definitive and fish intermediate hosts in Manabí province, Ecuador
    Article Snippet: .. For PCR-RFLP, we used the restriction enzyme Hind III (New England Biolabs, Ipswich, Massachusetts, United States), which targets a specific region of ITS2. .. The enzyme digestion was performed using 10 μl of PCR amplicons with 5 U of Hind III for one hour at 37°C.

    Article Title: Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases
    Article Snippet: .. The PCR products were digested with BamHI and HindIII restriction endonucleases (New England Biolabs, Ipswich, MA) and ligated into a pFastBac1 vector (Invitrogen, Carlsbad, CA) generating pFB-His-hDNA2-FLAG. .. The D277A point mutation inactivating the hDNA2 nuclease was introduced with oligonucleotide pair 5'-GGCCTGAAGGGAAAGATCGCTGTCACAGTTGGAGTGAAG-3' and 5'-CTTCACTCCAACTGTGACAGCGATCTTTCCCTTCAGGCC-3' whereas the K654R point mutation abolishing the hDNA2 helicase was introduced with oligonucleotide pair 5'-GGCATGCCGGGAACTGGCAGGACAACCACTATCTGCACA-3' and 5'-TGTGCAGATAGTGGTTGTCCTGCCAGTTCCCGGCATGCC-3' using the QuikChange XL Site-directed mutagenesis kit (Agilent, Santa Clara, CA) according to manufacturer's recommendations.

    Isolation:

    Article Title: Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma
    Article Snippet: .. Enzymatic treatment of nucleic acids isolated from IBsNucleic acids extracted from purified IBs were treated with RNAse A (Thermo Scientific™, 10 mg/ml), RNase T1 (Thermo Scientific™, 1000 U/ml), DNAse I, restriction endonucleases XhoI and HindIII (New England BioLabs) and Proteinase K (Roche, 10 μg/ml) following the manufacturers’ protocols. .. Isolation of RNA from hIFNγ IBs RNA was extracted from purified IBs by TRIzol® reagent (Invitrogen™) following the manufacturer’s protocol.

    Agarose Gel Electrophoresis:

    Article Title: GES Extended-Spectrum ?-Lactamases in Acinetobacter baumannii Isolates in Belgium ▿
    Article Snippet: .. One microgram of natural plasmids was digested with 10 U of HindIII and 10 U of EcoRI (New England Biolabs, Hitchin, United Kingdom) at 37°C for 1 h. Restricted fragments were separated on a 0.8% agarose gel in 0.5× Tris-borate-EDTA (TBE) (44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA, pH 8.3) buffer containing 0.5 μg/ml of ethidium bromide for 1 h at 150 V and visualized under UV light. ..

    Article Title: Emergence of Resistance among USA300 Methicillin-Resistant Staphylococcus aureus Isolates Causing Invasive Disease in the United States ▿
    Article Snippet: .. HindIII (New England Biolabs, Beverly, MA)-restricted plasmid fragments were separated on a 0.75% agarose gel in Tris-borate-EDTA (TBE) buffer at 80 V for 5 h. The Trackit 1 Kb DNA ladder (Invitrogen, Carlsbad, CA) was used for sizing the linear fragments. .. Digoxigenin-labeled DNA probes were generated by PCR using the oligonucleotide primers shown in Table and plasmid DNA isolated from the following control organisms: S. aureus FPR3757 ( ) for tra E , tra I , rep A , and mup A , and S. aureus HIP11714 MI-1 (vancomycin resistant S. aureus [VRSA-1]) ( ) for aac 6 ′- aph 2 ″ and dfr A .

    Plasmid Preparation:

    Article Title: Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases
    Article Snippet: .. The PCR products were digested with BamHI and HindIII restriction endonucleases (New England Biolabs, Ipswich, MA) and ligated into a pFastBac1 vector (Invitrogen, Carlsbad, CA) generating pFB-His-hDNA2-FLAG. .. The D277A point mutation inactivating the hDNA2 nuclease was introduced with oligonucleotide pair 5'-GGCCTGAAGGGAAAGATCGCTGTCACAGTTGGAGTGAAG-3' and 5'-CTTCACTCCAACTGTGACAGCGATCTTTCCCTTCAGGCC-3' whereas the K654R point mutation abolishing the hDNA2 helicase was introduced with oligonucleotide pair 5'-GGCATGCCGGGAACTGGCAGGACAACCACTATCTGCACA-3' and 5'-TGTGCAGATAGTGGTTGTCCTGCCAGTTCCCGGCATGCC-3' using the QuikChange XL Site-directed mutagenesis kit (Agilent, Santa Clara, CA) according to manufacturer's recommendations.

    Article Title: Emergence of Resistance among USA300 Methicillin-Resistant Staphylococcus aureus Isolates Causing Invasive Disease in the United States ▿
    Article Snippet: .. HindIII (New England Biolabs, Beverly, MA)-restricted plasmid fragments were separated on a 0.75% agarose gel in Tris-borate-EDTA (TBE) buffer at 80 V for 5 h. The Trackit 1 Kb DNA ladder (Invitrogen, Carlsbad, CA) was used for sizing the linear fragments. .. Digoxigenin-labeled DNA probes were generated by PCR using the oligonucleotide primers shown in Table and plasmid DNA isolated from the following control organisms: S. aureus FPR3757 ( ) for tra E , tra I , rep A , and mup A , and S. aureus HIP11714 MI-1 (vancomycin resistant S. aureus [VRSA-1]) ( ) for aac 6 ′- aph 2 ″ and dfr A .

    Purification:

    Article Title: Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma
    Article Snippet: .. Enzymatic treatment of nucleic acids isolated from IBsNucleic acids extracted from purified IBs were treated with RNAse A (Thermo Scientific™, 10 mg/ml), RNase T1 (Thermo Scientific™, 1000 U/ml), DNAse I, restriction endonucleases XhoI and HindIII (New England BioLabs) and Proteinase K (Roche, 10 μg/ml) following the manufacturers’ protocols. .. Isolation of RNA from hIFNγ IBs RNA was extracted from purified IBs by TRIzol® reagent (Invitrogen™) following the manufacturer’s protocol.

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    New England Biolabs hindiii restriction endonucleases
    hDNA2 D277A unwinds plasmid- and oligonucleotide-based DNA substrates. ( A ) Representative 1% agarose gel showing hDNA2 D277A helicase activity on a <t>λDNA/HindIII</t> substrate in a time-course experiment with 346 nM hRPA. Heat, heat-denatured DNA substrate. ( B ) Representative 1% agarose gel showing that nuclease- and helicase-deficient hDNA2 D277A K654R (lanes 2–6) and helicase-deficient hDNA2 K654R (lane 8) do not exhibit helicase activity. Lane 7, DNA unwinding by nuclease-deficient DNA2 D277A. Reactions contained 215 nM hRPA. ( C – E ) Representative 10% polyacrylamide gels showing the helicase activity of hDNA2 D277A with ( C ) 5’ overhang, ( D ) 3’ overhang and with ( E ) dsDNA substrates. Reactions contained 7.5 nM RPA. Heat, heat-denatured DNA substrate. ( F ) Representative 1% agarose gels showing DNA unwinding of a 2.7 kbp-long substrate by either hDNA2 D277A (left part, at 37°C) or yDna2 E675A (right part, at 30°C) in a kinetic experiment with 215 nM human RPA or 267 nM yeast RPA respectively. ( G ) Quantitation of experiments such as shown in F. Averages shown, n = 2; error bars, SEM. DOI: http://dx.doi.org/10.7554/eLife.18574.007
    Hindiii Restriction Endonucleases, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 799 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hDNA2 D277A unwinds plasmid- and oligonucleotide-based DNA substrates. ( A ) Representative 1% agarose gel showing hDNA2 D277A helicase activity on a λDNA/HindIII substrate in a time-course experiment with 346 nM hRPA. Heat, heat-denatured DNA substrate. ( B ) Representative 1% agarose gel showing that nuclease- and helicase-deficient hDNA2 D277A K654R (lanes 2–6) and helicase-deficient hDNA2 K654R (lane 8) do not exhibit helicase activity. Lane 7, DNA unwinding by nuclease-deficient DNA2 D277A. Reactions contained 215 nM hRPA. ( C – E ) Representative 10% polyacrylamide gels showing the helicase activity of hDNA2 D277A with ( C ) 5’ overhang, ( D ) 3’ overhang and with ( E ) dsDNA substrates. Reactions contained 7.5 nM RPA. Heat, heat-denatured DNA substrate. ( F ) Representative 1% agarose gels showing DNA unwinding of a 2.7 kbp-long substrate by either hDNA2 D277A (left part, at 37°C) or yDna2 E675A (right part, at 30°C) in a kinetic experiment with 215 nM human RPA or 267 nM yeast RPA respectively. ( G ) Quantitation of experiments such as shown in F. Averages shown, n = 2; error bars, SEM. DOI: http://dx.doi.org/10.7554/eLife.18574.007

    Journal: eLife

    Article Title: Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases

    doi: 10.7554/eLife.18574

    Figure Lengend Snippet: hDNA2 D277A unwinds plasmid- and oligonucleotide-based DNA substrates. ( A ) Representative 1% agarose gel showing hDNA2 D277A helicase activity on a λDNA/HindIII substrate in a time-course experiment with 346 nM hRPA. Heat, heat-denatured DNA substrate. ( B ) Representative 1% agarose gel showing that nuclease- and helicase-deficient hDNA2 D277A K654R (lanes 2–6) and helicase-deficient hDNA2 K654R (lane 8) do not exhibit helicase activity. Lane 7, DNA unwinding by nuclease-deficient DNA2 D277A. Reactions contained 215 nM hRPA. ( C – E ) Representative 10% polyacrylamide gels showing the helicase activity of hDNA2 D277A with ( C ) 5’ overhang, ( D ) 3’ overhang and with ( E ) dsDNA substrates. Reactions contained 7.5 nM RPA. Heat, heat-denatured DNA substrate. ( F ) Representative 1% agarose gels showing DNA unwinding of a 2.7 kbp-long substrate by either hDNA2 D277A (left part, at 37°C) or yDna2 E675A (right part, at 30°C) in a kinetic experiment with 215 nM human RPA or 267 nM yeast RPA respectively. ( G ) Quantitation of experiments such as shown in F. Averages shown, n = 2; error bars, SEM. DOI: http://dx.doi.org/10.7554/eLife.18574.007

    Article Snippet: The PCR products were digested with BamHI and HindIII restriction endonucleases (New England Biolabs, Ipswich, MA) and ligated into a pFastBac1 vector (Invitrogen, Carlsbad, CA) generating pFB-His-hDNA2-FLAG.

    Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Activity Assay, Recombinase Polymerase Amplification, Quantitation Assay

    a Agarose gel-electrophoresis of nucleic acids isolated from purified hIFNγ IBs. 1 —Sample, isolated from IBs, b Agarose gel-electrophoresis of nucleic acids isolated from purified hIFNγ IBs treated with restriction endonuclease XhoI and c HindIII. 1 —Non-treated sample; 2(b) —Sample treated with XhoI; 2(c) —sample treated with HindIII; d Enzymatic digestion of nucleic acids isolated from purified hIFNγ IBs 1 —non treated sample; 2 —sample treated with mixture of RNase A and RNase T 1 ; 3 —sample treated with DNase I; 4 —sample treated with Proteinase K; M —Molecular weight marker, bp

    Journal: Microbial Cell Factories

    Article Title: Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma

    doi: 10.1186/s12934-020-01400-6

    Figure Lengend Snippet: a Agarose gel-electrophoresis of nucleic acids isolated from purified hIFNγ IBs. 1 —Sample, isolated from IBs, b Agarose gel-electrophoresis of nucleic acids isolated from purified hIFNγ IBs treated with restriction endonuclease XhoI and c HindIII. 1 —Non-treated sample; 2(b) —Sample treated with XhoI; 2(c) —sample treated with HindIII; d Enzymatic digestion of nucleic acids isolated from purified hIFNγ IBs 1 —non treated sample; 2 —sample treated with mixture of RNase A and RNase T 1 ; 3 —sample treated with DNase I; 4 —sample treated with Proteinase K; M —Molecular weight marker, bp

    Article Snippet: Enzymatic treatment of nucleic acids isolated from IBsNucleic acids extracted from purified IBs were treated with RNAse A (Thermo Scientific™, 10 mg/ml), RNase T1 (Thermo Scientific™, 1000 U/ml), DNAse I, restriction endonucleases XhoI and HindIII (New England BioLabs) and Proteinase K (Roche, 10 μg/ml) following the manufacturers’ protocols.

    Techniques: Agarose Gel Electrophoresis, Isolation, Purification, Molecular Weight, Marker

    Molecular diagnosis of Amphimerus sp. metacercariae based on PCR-RFLP. (A) Multiple metacercariae were isolated from fish collected at each community. (B) Different metacercariae identified based on PCR-RFLP as shown in panel D. (C) First round of PCR amplification using universal trematode primers (green arrow). (D) Digested (L1-L6) and undigested (L7) PCR products by the restriction enzyme Hind III. Lanes 1–5 corresponds to (L1) adult Amphimerus sp. flukes from a human case at Esmeraldas, followed by adult parasites from a human (L2), a dog (L3), and a cat (L4), and (L5) Amphimerus sp. metacercariae from freshwater fish (i.e., Bryconamericus bucay ) from Pedro Pablo Gómez; the two fragments correspond to 374 and 140 base pairs (red arrows). Lane 6 corresponds to the metacercariae of Haplorchis pumilio with fragments of 440 and 86 base pairs (blue arrows). Lane 7 corresponds to unidentified metacercariae from fish in Pedro Pablo Gómez. L: 100 base pairs DNA Ladder (New England Biolabs, Ipswich, Massachusetts, United States).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Liver fluke infections by Amphimerus sp. (Digenea: Opisthorchiidae) in definitive and fish intermediate hosts in Manabí province, Ecuador

    doi: 10.1371/journal.pntd.0008286

    Figure Lengend Snippet: Molecular diagnosis of Amphimerus sp. metacercariae based on PCR-RFLP. (A) Multiple metacercariae were isolated from fish collected at each community. (B) Different metacercariae identified based on PCR-RFLP as shown in panel D. (C) First round of PCR amplification using universal trematode primers (green arrow). (D) Digested (L1-L6) and undigested (L7) PCR products by the restriction enzyme Hind III. Lanes 1–5 corresponds to (L1) adult Amphimerus sp. flukes from a human case at Esmeraldas, followed by adult parasites from a human (L2), a dog (L3), and a cat (L4), and (L5) Amphimerus sp. metacercariae from freshwater fish (i.e., Bryconamericus bucay ) from Pedro Pablo Gómez; the two fragments correspond to 374 and 140 base pairs (red arrows). Lane 6 corresponds to the metacercariae of Haplorchis pumilio with fragments of 440 and 86 base pairs (blue arrows). Lane 7 corresponds to unidentified metacercariae from fish in Pedro Pablo Gómez. L: 100 base pairs DNA Ladder (New England Biolabs, Ipswich, Massachusetts, United States).

    Article Snippet: For PCR-RFLP, we used the restriction enzyme Hind III (New England Biolabs, Ipswich, Massachusetts, United States), which targets a specific region of ITS2.

    Techniques: Polymerase Chain Reaction, Isolation, Fluorescence In Situ Hybridization, Amplification

    Crude plasmid extracts and banding patterns of plasmids pGES-11 and pGES-12 corestricted with HindIII and EcoRI. Lane M, 2 log DNA ladder (0.1 to 10.0 kb); lane 1, pGES-11 (from isolate 9027) extract digested with EcoRI and HindIII; lane 2, pGES-12 (from

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: GES Extended-Spectrum ?-Lactamases in Acinetobacter baumannii Isolates in Belgium ▿

    doi: 10.1128/AAC.00871-10

    Figure Lengend Snippet: Crude plasmid extracts and banding patterns of plasmids pGES-11 and pGES-12 corestricted with HindIII and EcoRI. Lane M, 2 log DNA ladder (0.1 to 10.0 kb); lane 1, pGES-11 (from isolate 9027) extract digested with EcoRI and HindIII; lane 2, pGES-12 (from

    Article Snippet: One microgram of natural plasmids was digested with 10 U of HindIII and 10 U of EcoRI (New England Biolabs, Hitchin, United Kingdom) at 37°C for 1 h. Restricted fragments were separated on a 0.8% agarose gel in 0.5× Tris-borate-EDTA (TBE) (44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA, pH 8.3) buffer containing 0.5 μg/ml of ethidium bromide for 1 h at 150 V and visualized under UV light.

    Techniques: Plasmid Preparation