exonuclease iii  (Thermo Fisher)


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  • 99
    Name:
    Exonuclease III 200 U µL
    Description:
    Thermo Scientific Exonuclease III ExoIII exhibits four catalytic activities The 3 →5 exodeoxyribonuclease activity of ExoIII is specific for double stranded DNA ExoIII degrades dsDNA from blunt ends 5 overhangs or nicks releases 5 mononucleotides from the 3 ends of DNA strands and produces stretches of single stranded DNA It is not active on 3 overhang ends of DNA that are at least four bases long and do not carry a 3 terminal C residue on single stranded DNA or on phosphorothioate linked nucleotides ExoIII 3 phosphatase activity removes the 3 terminal phosphate generating a 3 OH group ExoIII Rnase H activity exonucleolytically degrades the RNA strand in RNA DNA hybrids ExoIII apurinic apyrimidinic endonuclease activity cleaves phosphodiester bonds at apurinic or apyrimidinic sites to produce 5 termini that are base free deoxyribose 5 phosphate residues Highlights• Active in restriction enzyme buffersApplications• Creation of unidirectional deletions in DNA fragments in conjunction with S1 Nuclease• Generation of a single stranded template for dideoxy sequencing of DNA• Site directed mutagenesis• Cloning of PCR products• Preparation of strand specific probesNoteThe rate of DNA digestion by ExoIII depends upon temperature salt concentration and the molar ratio of DNA to enzyme in the reaction mixture Optimal reaction conditions should be determined experimentally
    Catalog Number:
    en0191
    Price:
    None
    Applications:
    Cloning|PCR Cloning|Mutagenesis
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher exonuclease iii
    Evidence of a covalently linked 5′ TP. (A) <t>DNA-protein</t> complex (lane 1), complex treated with exonuclease <t>III</t> for 15 min (lane 2), complex treated with exonuclease III for 30 min (lane 3), PK-DNA (lane 4), PK-DNA treated with exonuclease III for 15 min (lane 5), and PK-DNA treated with exonuclease III for 30 min (lane 6). (B) DNA-protein complex (lane 1), complex treated with λ exonuclease for 15 min (lane 2), complex treated with λ exonuclease for 30 min (lane 3), PK-DNA (lane 4), PK-DNA treated with λ exonuclease for 30 min (lane 5), PK-DNA treated with 0.5 M piperidine (lane 6), PK-DNA treated with piperidine and λ exonuclease for 30 min (lane 7). The amounts used are indicated in Materials and Methods.
    Thermo Scientific Exonuclease III ExoIII exhibits four catalytic activities The 3 →5 exodeoxyribonuclease activity of ExoIII is specific for double stranded DNA ExoIII degrades dsDNA from blunt ends 5 overhangs or nicks releases 5 mononucleotides from the 3 ends of DNA strands and produces stretches of single stranded DNA It is not active on 3 overhang ends of DNA that are at least four bases long and do not carry a 3 terminal C residue on single stranded DNA or on phosphorothioate linked nucleotides ExoIII 3 phosphatase activity removes the 3 terminal phosphate generating a 3 OH group ExoIII Rnase H activity exonucleolytically degrades the RNA strand in RNA DNA hybrids ExoIII apurinic apyrimidinic endonuclease activity cleaves phosphodiester bonds at apurinic or apyrimidinic sites to produce 5 termini that are base free deoxyribose 5 phosphate residues Highlights• Active in restriction enzyme buffersApplications• Creation of unidirectional deletions in DNA fragments in conjunction with S1 Nuclease• Generation of a single stranded template for dideoxy sequencing of DNA• Site directed mutagenesis• Cloning of PCR products• Preparation of strand specific probesNoteThe rate of DNA digestion by ExoIII depends upon temperature salt concentration and the molar ratio of DNA to enzyme in the reaction mixture Optimal reaction conditions should be determined experimentally
    https://www.bioz.com/result/exonuclease iii/product/Thermo Fisher
    Average 99 stars, based on 26 article reviews
    Price from $9.99 to $1999.99
    exonuclease iii - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Genomic Sequence of C1, the First Streptococcal Phage"

    Article Title: Genomic Sequence of C1, the First Streptococcal Phage

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.185.11.3325-3332.2003

    Evidence of a covalently linked 5′ TP. (A) DNA-protein complex (lane 1), complex treated with exonuclease III for 15 min (lane 2), complex treated with exonuclease III for 30 min (lane 3), PK-DNA (lane 4), PK-DNA treated with exonuclease III for 15 min (lane 5), and PK-DNA treated with exonuclease III for 30 min (lane 6). (B) DNA-protein complex (lane 1), complex treated with λ exonuclease for 15 min (lane 2), complex treated with λ exonuclease for 30 min (lane 3), PK-DNA (lane 4), PK-DNA treated with λ exonuclease for 30 min (lane 5), PK-DNA treated with 0.5 M piperidine (lane 6), PK-DNA treated with piperidine and λ exonuclease for 30 min (lane 7). The amounts used are indicated in Materials and Methods.
    Figure Legend Snippet: Evidence of a covalently linked 5′ TP. (A) DNA-protein complex (lane 1), complex treated with exonuclease III for 15 min (lane 2), complex treated with exonuclease III for 30 min (lane 3), PK-DNA (lane 4), PK-DNA treated with exonuclease III for 15 min (lane 5), and PK-DNA treated with exonuclease III for 30 min (lane 6). (B) DNA-protein complex (lane 1), complex treated with λ exonuclease for 15 min (lane 2), complex treated with λ exonuclease for 30 min (lane 3), PK-DNA (lane 4), PK-DNA treated with λ exonuclease for 30 min (lane 5), PK-DNA treated with 0.5 M piperidine (lane 6), PK-DNA treated with piperidine and λ exonuclease for 30 min (lane 7). The amounts used are indicated in Materials and Methods.

    Techniques Used:

    2) Product Images from "DIRS retrotransposons amplify via linear, single-stranded cDNA intermediates"

    Article Title: DIRS retrotransposons amplify via linear, single-stranded cDNA intermediates

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa160

    Features of extrachromosomal DIRS-1 bsr cDNA. Extrachromosomal cDNA samples were extracted from rrpC– strains transformed with DIRS-1 bsr ( A ) or DIRS-1 bsr* ( B ), and were cultivated in G418/BS10 medium that selects for strains with mobilized retrotransposon. The samples were treated separately with RNase A, DNase I, Exonuclease I (Exo I), Exonuclease III (Exo III) and S1 nuclease (S1). After digestion and heat inactivation of enzymes, the samples were analyzed by PCR with the primers #2212 and #2213 binding within the region of mbsrI cassette (binding position in Figure 2A ). Quantification of endogenous ( C and D ) and mbsrI -tagged versions ( E ) of DIRS-1 extrachromosomal cDNA after treatment with Exonuclease I and S1 nuclease, relative to the non-digested sample. Samples were extracted from rrpC– strains transformed with DIRS-1 bsr and DIRS-1 bsr* upon cultivation in G418/BS10 medium that selects for strains with mobilized retrotransposons. The cDNA abundance was monitored by qPCR using primers targeting positions qP1, qP2 and qBS (Figures 2A and 3A ) and is shown in logarithmic scale relative to the non-digested sample (set to 1). Each qPCR reaction was run in triplicate. Error bars : mean with S.D. Statistics: paired t test: P
    Figure Legend Snippet: Features of extrachromosomal DIRS-1 bsr cDNA. Extrachromosomal cDNA samples were extracted from rrpC– strains transformed with DIRS-1 bsr ( A ) or DIRS-1 bsr* ( B ), and were cultivated in G418/BS10 medium that selects for strains with mobilized retrotransposon. The samples were treated separately with RNase A, DNase I, Exonuclease I (Exo I), Exonuclease III (Exo III) and S1 nuclease (S1). After digestion and heat inactivation of enzymes, the samples were analyzed by PCR with the primers #2212 and #2213 binding within the region of mbsrI cassette (binding position in Figure 2A ). Quantification of endogenous ( C and D ) and mbsrI -tagged versions ( E ) of DIRS-1 extrachromosomal cDNA after treatment with Exonuclease I and S1 nuclease, relative to the non-digested sample. Samples were extracted from rrpC– strains transformed with DIRS-1 bsr and DIRS-1 bsr* upon cultivation in G418/BS10 medium that selects for strains with mobilized retrotransposons. The cDNA abundance was monitored by qPCR using primers targeting positions qP1, qP2 and qBS (Figures 2A and 3A ) and is shown in logarithmic scale relative to the non-digested sample (set to 1). Each qPCR reaction was run in triplicate. Error bars : mean with S.D. Statistics: paired t test: P

    Techniques Used: Transformation Assay, Polymerase Chain Reaction, Binding Assay, Real-time Polymerase Chain Reaction

    3) Product Images from "Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma"

    Article Title: Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma

    Journal: Journal of Extracellular Vesicles

    doi: 10.1080/20013078.2018.1505403

    Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells. NP4000 membrane, which can detect particles with a diameter between 1.0 and 6.0 μm, was used for quantitation of L-EVs, while NP200 membrane, which can detect particles with a diameter between 60 and 400 nm, was used for quantitation of S-EVs. (b) Protein lysates from L-EVs and S-EVs purified by iodixanol density gradient (at 1.10 and 1.15 g/ml) were blotted with LO markers HSPA5 and CK18, and with Exo marker CD81. (c) Total DNA was quantified by Qubit Fluorometer in L-EVs and S-EVs isolated from PC3 and U87 cell lines. The plot shows the DNA ratio between L-EVs and S-EVs. (d) Double stranded (ds)DNA was quantified by High Sensitivity (HS) dsDNA Qubit Assay in L-EVs and S-EVs isolated from 1 ml of plasma from patients with mCRPC ( n = 40) and cancer-free individuals ( n = 6). (e) Quantification of both protein and DNA content in L-EVs and S-EVs isolated from conditioned media of 12.6 × 10 7 PC3 cells. (f) Single stranded (ss) and dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Exonuclease III, were quantified by Qubit. (g) Chip-based capillary electrophoresis (Bioanalyzer) showing the presence of dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Endonuclease III. L-EVs contain abundant DNA with a large peak around 10 kbp. Conversely, the amount of DNA in S-EVs is negligible. (h) ss- and dsDNA in PC3-derived L-EVs and S-EVs were quantified by Qubit after treatment with nucleases (DNase I and Exonuclease III) with or without addition of a detergent (Triton X-100) prior to nuclease treatment. (i) Chip-based capillary electrophoresis (Bioanalyzer) showing that only miniscule amounts of dsDNA could be detected after EV lysis using a detergent prior to treatment with nucleases.
    Figure Legend Snippet: Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells. NP4000 membrane, which can detect particles with a diameter between 1.0 and 6.0 μm, was used for quantitation of L-EVs, while NP200 membrane, which can detect particles with a diameter between 60 and 400 nm, was used for quantitation of S-EVs. (b) Protein lysates from L-EVs and S-EVs purified by iodixanol density gradient (at 1.10 and 1.15 g/ml) were blotted with LO markers HSPA5 and CK18, and with Exo marker CD81. (c) Total DNA was quantified by Qubit Fluorometer in L-EVs and S-EVs isolated from PC3 and U87 cell lines. The plot shows the DNA ratio between L-EVs and S-EVs. (d) Double stranded (ds)DNA was quantified by High Sensitivity (HS) dsDNA Qubit Assay in L-EVs and S-EVs isolated from 1 ml of plasma from patients with mCRPC ( n = 40) and cancer-free individuals ( n = 6). (e) Quantification of both protein and DNA content in L-EVs and S-EVs isolated from conditioned media of 12.6 × 10 7 PC3 cells. (f) Single stranded (ss) and dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Exonuclease III, were quantified by Qubit. (g) Chip-based capillary electrophoresis (Bioanalyzer) showing the presence of dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Endonuclease III. L-EVs contain abundant DNA with a large peak around 10 kbp. Conversely, the amount of DNA in S-EVs is negligible. (h) ss- and dsDNA in PC3-derived L-EVs and S-EVs were quantified by Qubit after treatment with nucleases (DNase I and Exonuclease III) with or without addition of a detergent (Triton X-100) prior to nuclease treatment. (i) Chip-based capillary electrophoresis (Bioanalyzer) showing that only miniscule amounts of dsDNA could be detected after EV lysis using a detergent prior to treatment with nucleases.

    Techniques Used: Tunable Resistive Pulse Sensing, Derivative Assay, Quantitation Assay, Purification, Marker, Isolation, HS DSDNA Qubit Assay, Chromatin Immunoprecipitation, Electrophoresis, Lysis

    4) Product Images from "Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences"

    Article Title: Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences

    Journal: Nature communications

    doi: 10.1038/ncomms1180

    Medulloblastomas with amplified c-Myc oncogenes have elevated c-Myc exoRNA and exoDNA in their microvesicles ( a ) c-Myc amplification levels were quantified in genomic DNA (gDNA) from one normal human fibroblast line (HF19), one GBM line (11/5), one atypical teratoid rhabdoid tumour (AT/RT) line (NS224) and three medulloblastoma (MB) lines (D425, D458 and D384). ExoRNA and exoDNA were also isolated from their corresponding microvesicles. ( b ) qRT-PCR and ( c ) qPCR were carried out on nucleic acid from microvesicles from the same cell lines to measure exoRNA and exoDNA, respectively. c-Myc levels were normalized to GAPDH in the same preparation and expressed as fold increase relative to normal fibroblasts. In all cases, values are expressed as mean ± s.e.m. ( n = 3) and analysed by two-tailed t -test comparing MB lines to HF19 (* P
    Figure Legend Snippet: Medulloblastomas with amplified c-Myc oncogenes have elevated c-Myc exoRNA and exoDNA in their microvesicles ( a ) c-Myc amplification levels were quantified in genomic DNA (gDNA) from one normal human fibroblast line (HF19), one GBM line (11/5), one atypical teratoid rhabdoid tumour (AT/RT) line (NS224) and three medulloblastoma (MB) lines (D425, D458 and D384). ExoRNA and exoDNA were also isolated from their corresponding microvesicles. ( b ) qRT-PCR and ( c ) qPCR were carried out on nucleic acid from microvesicles from the same cell lines to measure exoRNA and exoDNA, respectively. c-Myc levels were normalized to GAPDH in the same preparation and expressed as fold increase relative to normal fibroblasts. In all cases, values are expressed as mean ± s.e.m. ( n = 3) and analysed by two-tailed t -test comparing MB lines to HF19 (* P

    Techniques Used: Amplification, Isolation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Two Tailed Test

    Retrotransposon DNA sequences in microvesicles Cellular and microvesicle DNA was isolated from three medulloblastoma (D425, D384 and D458), one GBM (11/5), one melanoma (0106) and one human fibroblast (HF19) line. qPCR analysis was carried out for ( a ) L1, ( b ) Alu and ( c ) HERV-K in gDNA and exoDNA. Ct values were normalized to GAPDH levels and are shown as relative enrichment of transposons in cells ( y
    Figure Legend Snippet: Retrotransposon DNA sequences in microvesicles Cellular and microvesicle DNA was isolated from three medulloblastoma (D425, D384 and D458), one GBM (11/5), one melanoma (0106) and one human fibroblast (HF19) line. qPCR analysis was carried out for ( a ) L1, ( b ) Alu and ( c ) HERV-K in gDNA and exoDNA. Ct values were normalized to GAPDH levels and are shown as relative enrichment of transposons in cells ( y

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction

    5) Product Images from "Argonaute Proteins Affect siRNA Levels and Accumulation of a Novel Extrachromosomal DNA from the Dictyostelium Retrotransposon DIRS-1 *"

    Article Title: Argonaute Proteins Affect siRNA Levels and Accumulation of a Novel Extrachromosomal DNA from the Dictyostelium Retrotransposon DIRS-1 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.612663

    Expression of DIRS-1 transcripts in different D. discoideum knock-out strains. A ) consisting of long inverted terminal repeats ( left ITR (330 bp) and right ITR (358 bp)) that flank three open reading frames. ORF1 encodes a putative Gag protein, ORF2 encodes a tyrosine recombinase ( YR ), and ORF3 encodes the pol gene, which comprises a RT, an RNase H ( RH ), and a methyltransferase ( MT ) domain. The internal complementary region ( ICR ) next to the right ITR exhibits sequences that are inverse complementary to the left and the right ITRs. A1 and A2 indicate amplicons used for qRT-PCR. P1 to P3 show the binding positions of the radioactively labeled oligonucleotides. Oligonucleotide probes ( P ) shown above the element are directed against sense transcripts. B , Northern blot analysis of DIRS-1 sense transcripts in deletion strains and the Ax2 wild type. Three 32 P-labeled oligonucleotides (P1–P3) were used to detect sense transcripts. As a loading control, the membrane was rehybridized with a 32 P-labeled oligonucleotide probe directed against the actin mRNA. C , relative expression of DIRS-1 transcripts (sense and antisense) in the indicated deletion strains and the Ax2 wild type measured by qRT-PCR using two different DIRS-1 amplicons ( A1 and A2 ) normalized to cinD mRNA. The data were plotted relative to DIRS-1 expression in Ax2 (transcript level is set to 100%). A1 , n = 8. Error bars : mean with S.D.; paired t test: p = 0.0001 (***) ( Ax2 wt : agnA−), p = 0.0037 (**) ( Ax2 : agnB−), p = 0.0010 (***) (Ax2: agnA−/agnB−). A2 , n = 4 (agnA−/agnB ko n = 4). Error bars : mean with S.D., paired t test: p =
    Figure Legend Snippet: Expression of DIRS-1 transcripts in different D. discoideum knock-out strains. A ) consisting of long inverted terminal repeats ( left ITR (330 bp) and right ITR (358 bp)) that flank three open reading frames. ORF1 encodes a putative Gag protein, ORF2 encodes a tyrosine recombinase ( YR ), and ORF3 encodes the pol gene, which comprises a RT, an RNase H ( RH ), and a methyltransferase ( MT ) domain. The internal complementary region ( ICR ) next to the right ITR exhibits sequences that are inverse complementary to the left and the right ITRs. A1 and A2 indicate amplicons used for qRT-PCR. P1 to P3 show the binding positions of the radioactively labeled oligonucleotides. Oligonucleotide probes ( P ) shown above the element are directed against sense transcripts. B , Northern blot analysis of DIRS-1 sense transcripts in deletion strains and the Ax2 wild type. Three 32 P-labeled oligonucleotides (P1–P3) were used to detect sense transcripts. As a loading control, the membrane was rehybridized with a 32 P-labeled oligonucleotide probe directed against the actin mRNA. C , relative expression of DIRS-1 transcripts (sense and antisense) in the indicated deletion strains and the Ax2 wild type measured by qRT-PCR using two different DIRS-1 amplicons ( A1 and A2 ) normalized to cinD mRNA. The data were plotted relative to DIRS-1 expression in Ax2 (transcript level is set to 100%). A1 , n = 8. Error bars : mean with S.D.; paired t test: p = 0.0001 (***) ( Ax2 wt : agnA−), p = 0.0037 (**) ( Ax2 : agnB−), p = 0.0010 (***) (Ax2: agnA−/agnB−). A2 , n = 4 (agnA−/agnB ko n = 4). Error bars : mean with S.D., paired t test: p =

    Techniques Used: Expressing, Knock-Out, Quantitative RT-PCR, Binding Assay, Labeling, Northern Blot

    6) Product Images from "Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ"

    Article Title: Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052584

    Copper(I) treatment produces short gaps with phosphate groups at the 3′ end. A ) TdT was used to incorporate Alexa-dUTP at the 3′ end of the gaps. A strong signal is observed only after the pre-incubation of cells with exonuclease III or SAP. The model shows the situation after the action of SAP in the case of double-stranded DNA with several gaps. Although the phosphate groups are shown also at the 5′ end of the gaps, it is not clear whether they are present there. Therefore, the action of SAP is shown for 3′ phosphate groups exclusively. Bar: 20 µm. B ) DNA polymerase I, Klenow fragment and Klenow fragment Exo- were used to incorporate Alexa-dUTP at the gap sites produced by monovalent copper. Only DNA polymerase I produced a strong signal. When incubation with exonuclease III preceded the polymerase step, a strong signal was observed also in the case of both Klenow fragments. The model shows the action of DNA polymerase I at the sites of created gaps. Both 3′-5′ proofreading activity enabling hydroxyl group formation and 5′-3′ exonuclease activity (for the sake of simplicity, the excised nucleotides are not shown in the model) enabling nick translation are necessary. As no ligase activity was present, nicks at the ends of the labeled chains persisted (arrows in the model picture), although it is not apparent. Bar: 20 µm.
    Figure Legend Snippet: Copper(I) treatment produces short gaps with phosphate groups at the 3′ end. A ) TdT was used to incorporate Alexa-dUTP at the 3′ end of the gaps. A strong signal is observed only after the pre-incubation of cells with exonuclease III or SAP. The model shows the situation after the action of SAP in the case of double-stranded DNA with several gaps. Although the phosphate groups are shown also at the 5′ end of the gaps, it is not clear whether they are present there. Therefore, the action of SAP is shown for 3′ phosphate groups exclusively. Bar: 20 µm. B ) DNA polymerase I, Klenow fragment and Klenow fragment Exo- were used to incorporate Alexa-dUTP at the gap sites produced by monovalent copper. Only DNA polymerase I produced a strong signal. When incubation with exonuclease III preceded the polymerase step, a strong signal was observed also in the case of both Klenow fragments. The model shows the action of DNA polymerase I at the sites of created gaps. Both 3′-5′ proofreading activity enabling hydroxyl group formation and 5′-3′ exonuclease activity (for the sake of simplicity, the excised nucleotides are not shown in the model) enabling nick translation are necessary. As no ligase activity was present, nicks at the ends of the labeled chains persisted (arrows in the model picture), although it is not apparent. Bar: 20 µm.

    Techniques Used: Incubation, Produced, Activity Assay, Nick Translation, Labeling

    Copper(I)-oxygen efficiently reveals incorporated BrdU; the revelation can be further increased by means of exonucleases. A ) The results of the detection of the BrdU labeling of replicated DNA using acid (4 N HCl) or hydroxide (0.07 M NaOH) or DNase I treatment or the one-step or the two-step procedure are shown. All of the images were taken using 99-ms time to be able to compare the signal intensity. In the one-step procedure (the image labeled as Cu), the 30-minute treatment with copper(I)-oxygen was used exclusively. In the two-step protocol, a 10-minute treatment of the samples with copper(I)-oxygen was followed by incubation with exonuclease III or exonuclease λ. The model shows the situation for both one-step and two-step procedures. Note that exonuclease λ reveals BrdU-labeled parts in the proximity of close single gaps as it has no activity at nicks and limited activity at gaps. Only close single gaps can result into the formation of double-strand break. Although only one strand is usually labeled by BrdU, the situation is shown as if both strands were labeled in the schematic picture. The revealed parts of distinct strands are distinguished by colors. Bar: 20 µm. B ) Relative signal intensity is shown in the graph.
    Figure Legend Snippet: Copper(I)-oxygen efficiently reveals incorporated BrdU; the revelation can be further increased by means of exonucleases. A ) The results of the detection of the BrdU labeling of replicated DNA using acid (4 N HCl) or hydroxide (0.07 M NaOH) or DNase I treatment or the one-step or the two-step procedure are shown. All of the images were taken using 99-ms time to be able to compare the signal intensity. In the one-step procedure (the image labeled as Cu), the 30-minute treatment with copper(I)-oxygen was used exclusively. In the two-step protocol, a 10-minute treatment of the samples with copper(I)-oxygen was followed by incubation with exonuclease III or exonuclease λ. The model shows the situation for both one-step and two-step procedures. Note that exonuclease λ reveals BrdU-labeled parts in the proximity of close single gaps as it has no activity at nicks and limited activity at gaps. Only close single gaps can result into the formation of double-strand break. Although only one strand is usually labeled by BrdU, the situation is shown as if both strands were labeled in the schematic picture. The revealed parts of distinct strands are distinguished by colors. Bar: 20 µm. B ) Relative signal intensity is shown in the graph.

    Techniques Used: Labeling, Mass Spectrometry, Incubation, Activity Assay

    Copper(I) treatment produces short gaps with phosphate groups at the 3′ end. A ) TdT was used to incorporate Alexa-dUTP at the 3′ end of the gaps. A strong signal is observed only after the pre-incubation of cells with exonuclease III or SAP. The model shows the situation after the action of SAP in the case of double-stranded DNA with several gaps. Although the phosphate groups are shown also at the 5′ end of the gaps, it is not clear whether they are present there. Therefore, the action of SAP is shown for 3′ phosphate groups exclusively. Bar: 20 µm. B ) DNA polymerase I, Klenow fragment and Klenow fragment Exo- were used to incorporate Alexa-dUTP at the gap sites produced by monovalent copper. Only DNA polymerase I produced a strong signal. When incubation with exonuclease III preceded the polymerase step, a strong signal was observed also in the case of both Klenow fragments. The model shows the action of DNA polymerase I at the sites of created gaps. Both 3′-5′ proofreading activity enabling hydroxyl group formation and 5′-3′ exonuclease activity (for the sake of simplicity, the excised nucleotides are not shown in the model) enabling nick translation are necessary. As no ligase activity was present, nicks at the ends of the labeled chains persisted (arrows in the model picture), although it is not apparent. Bar: 20 µm.
    Figure Legend Snippet: Copper(I) treatment produces short gaps with phosphate groups at the 3′ end. A ) TdT was used to incorporate Alexa-dUTP at the 3′ end of the gaps. A strong signal is observed only after the pre-incubation of cells with exonuclease III or SAP. The model shows the situation after the action of SAP in the case of double-stranded DNA with several gaps. Although the phosphate groups are shown also at the 5′ end of the gaps, it is not clear whether they are present there. Therefore, the action of SAP is shown for 3′ phosphate groups exclusively. Bar: 20 µm. B ) DNA polymerase I, Klenow fragment and Klenow fragment Exo- were used to incorporate Alexa-dUTP at the gap sites produced by monovalent copper. Only DNA polymerase I produced a strong signal. When incubation with exonuclease III preceded the polymerase step, a strong signal was observed also in the case of both Klenow fragments. The model shows the action of DNA polymerase I at the sites of created gaps. Both 3′-5′ proofreading activity enabling hydroxyl group formation and 5′-3′ exonuclease activity (for the sake of simplicity, the excised nucleotides are not shown in the model) enabling nick translation are necessary. As no ligase activity was present, nicks at the ends of the labeled chains persisted (arrows in the model picture), although it is not apparent. Bar: 20 µm.

    Techniques Used: Incubation, Produced, Activity Assay, Nick Translation, Labeling

    7) Product Images from "Quantitative Microplate Assay for Real-Time Nuclease Kinetics"

    Article Title: Quantitative Microplate Assay for Real-Time Nuclease Kinetics

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0154099

    Exonuclease III steady state kinetics with increasing concentration of substrate by phosphate release assay. 1.5 nM ExoIII was incubated with 1μM MDCC-PBP, 0.0004u/μl FastAP and varying concentrations (5, 10, 20, 40, 60, 100 and 200 nM) of dsDNA substrate in 66mM Tris-HCl (pH 8.0) and 0.66mM MgCl 2 at 37°C. (A) Fluorescence increase was measured over time from the ExoIII reaction coupled to FastAP dephosphorylation of products and subsequent P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S5 Fig ). Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of substrate in the different data sets. (B) Michaelis-Menten saturation curve by plotting initial velocity (v 0 ) obtained from (A) against 3’-end concentration. Constants derived from plot were V max = 0.5947 ± 0.0380 nM s -1 and K M = 140.9 ± 20.3 nM.
    Figure Legend Snippet: Exonuclease III steady state kinetics with increasing concentration of substrate by phosphate release assay. 1.5 nM ExoIII was incubated with 1μM MDCC-PBP, 0.0004u/μl FastAP and varying concentrations (5, 10, 20, 40, 60, 100 and 200 nM) of dsDNA substrate in 66mM Tris-HCl (pH 8.0) and 0.66mM MgCl 2 at 37°C. (A) Fluorescence increase was measured over time from the ExoIII reaction coupled to FastAP dephosphorylation of products and subsequent P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S5 Fig ). Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of substrate in the different data sets. (B) Michaelis-Menten saturation curve by plotting initial velocity (v 0 ) obtained from (A) against 3’-end concentration. Constants derived from plot were V max = 0.5947 ± 0.0380 nM s -1 and K M = 140.9 ± 20.3 nM.

    Techniques Used: Concentration Assay, Phosphate Release Assay, Incubation, Fluorescence, De-Phosphorylation Assay, Binding Assay, Derivative Assay

    Phosphate release assay for ‘10–23’ DNAzyme steady state kinetics. Reactions were set up containing 2 μM RNA substrate, 1 μM MDCC-PBP, 0.3u/μl T4PNK and varying concentrations (50, 100, 150 and 200 nM) of DzSJ in 40 mM Tris-HCl (pH 7.5), 20 mM MgCl2 and 50 mM NaCl at room temperature. As a control 200 nM inactive DzSJ was used instead of DzSJ. Cleavage of RNA substrate exposes a 2’, 3’-cyclic phosphate ( > P). The cyclic phosphate is released by T4PNK and quantified by fluorescence increase caused by P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S6 Fig ). (A) Inorganic phosphate concentration plotted over time. Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of DzSJ in the different data sets. (B) Initial velocities (v 0 ) obtained from (A) plotted against DzSJ concentration showing a linear relation. R 2 = 0.98.
    Figure Legend Snippet: Phosphate release assay for ‘10–23’ DNAzyme steady state kinetics. Reactions were set up containing 2 μM RNA substrate, 1 μM MDCC-PBP, 0.3u/μl T4PNK and varying concentrations (50, 100, 150 and 200 nM) of DzSJ in 40 mM Tris-HCl (pH 7.5), 20 mM MgCl2 and 50 mM NaCl at room temperature. As a control 200 nM inactive DzSJ was used instead of DzSJ. Cleavage of RNA substrate exposes a 2’, 3’-cyclic phosphate ( > P). The cyclic phosphate is released by T4PNK and quantified by fluorescence increase caused by P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S6 Fig ). (A) Inorganic phosphate concentration plotted over time. Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of DzSJ in the different data sets. (B) Initial velocities (v 0 ) obtained from (A) plotted against DzSJ concentration showing a linear relation. R 2 = 0.98.

    Techniques Used: Phosphate Release Assay, Fluorescence, Binding Assay, Concentration Assay

    8) Product Images from "Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ"

    Article Title: Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052584

    Copper(I) treatment produces short gaps with phosphate groups at the 3′ end. A ) TdT was used to incorporate Alexa-dUTP at the 3′ end of the gaps. A strong signal is observed only after the pre-incubation of cells with exonuclease III or SAP. The model shows the situation after the action of SAP in the case of double-stranded DNA with several gaps. Although the phosphate groups are shown also at the 5′ end of the gaps, it is not clear whether they are present there. Therefore, the action of SAP is shown for 3′ phosphate groups exclusively. Bar: 20 µm. B ) DNA polymerase I, Klenow fragment and Klenow fragment Exo- were used to incorporate Alexa-dUTP at the gap sites produced by monovalent copper. Only DNA polymerase I produced a strong signal. When incubation with exonuclease III preceded the polymerase step, a strong signal was observed also in the case of both Klenow fragments. The model shows the action of DNA polymerase I at the sites of created gaps. Both 3′-5′ proofreading activity enabling hydroxyl group formation and 5′-3′ exonuclease activity (for the sake of simplicity, the excised nucleotides are not shown in the model) enabling nick translation are necessary. As no ligase activity was present, nicks at the ends of the labeled chains persisted (arrows in the model picture), although it is not apparent. Bar: 20 µm.
    Figure Legend Snippet: Copper(I) treatment produces short gaps with phosphate groups at the 3′ end. A ) TdT was used to incorporate Alexa-dUTP at the 3′ end of the gaps. A strong signal is observed only after the pre-incubation of cells with exonuclease III or SAP. The model shows the situation after the action of SAP in the case of double-stranded DNA with several gaps. Although the phosphate groups are shown also at the 5′ end of the gaps, it is not clear whether they are present there. Therefore, the action of SAP is shown for 3′ phosphate groups exclusively. Bar: 20 µm. B ) DNA polymerase I, Klenow fragment and Klenow fragment Exo- were used to incorporate Alexa-dUTP at the gap sites produced by monovalent copper. Only DNA polymerase I produced a strong signal. When incubation with exonuclease III preceded the polymerase step, a strong signal was observed also in the case of both Klenow fragments. The model shows the action of DNA polymerase I at the sites of created gaps. Both 3′-5′ proofreading activity enabling hydroxyl group formation and 5′-3′ exonuclease activity (for the sake of simplicity, the excised nucleotides are not shown in the model) enabling nick translation are necessary. As no ligase activity was present, nicks at the ends of the labeled chains persisted (arrows in the model picture), although it is not apparent. Bar: 20 µm.

    Techniques Used: Incubation, Produced, Activity Assay, Nick Translation, Labeling

    Copper(I)-oxygen efficiently reveals incorporated BrdU; the revelation can be further increased by means of exonucleases. A ) The results of the detection of the BrdU labeling of replicated DNA using acid (4 N HCl) or hydroxide (0.07 M NaOH) or DNase I treatment or the one-step or the two-step procedure are shown. All of the images were taken using 99-ms time to be able to compare the signal intensity. In the one-step procedure (the image labeled as Cu), the 30-minute treatment with copper(I)-oxygen was used exclusively. In the two-step protocol, a 10-minute treatment of the samples with copper(I)-oxygen was followed by incubation with exonuclease III or exonuclease λ. The model shows the situation for both one-step and two-step procedures. Note that exonuclease λ reveals BrdU-labeled parts in the proximity of close single gaps as it has no activity at nicks and limited activity at gaps. Only close single gaps can result into the formation of double-strand break. Although only one strand is usually labeled by BrdU, the situation is shown as if both strands were labeled in the schematic picture. The revealed parts of distinct strands are distinguished by colors. Bar: 20 µm. B ) Relative signal intensity is shown in the graph.
    Figure Legend Snippet: Copper(I)-oxygen efficiently reveals incorporated BrdU; the revelation can be further increased by means of exonucleases. A ) The results of the detection of the BrdU labeling of replicated DNA using acid (4 N HCl) or hydroxide (0.07 M NaOH) or DNase I treatment or the one-step or the two-step procedure are shown. All of the images were taken using 99-ms time to be able to compare the signal intensity. In the one-step procedure (the image labeled as Cu), the 30-minute treatment with copper(I)-oxygen was used exclusively. In the two-step protocol, a 10-minute treatment of the samples with copper(I)-oxygen was followed by incubation with exonuclease III or exonuclease λ. The model shows the situation for both one-step and two-step procedures. Note that exonuclease λ reveals BrdU-labeled parts in the proximity of close single gaps as it has no activity at nicks and limited activity at gaps. Only close single gaps can result into the formation of double-strand break. Although only one strand is usually labeled by BrdU, the situation is shown as if both strands were labeled in the schematic picture. The revealed parts of distinct strands are distinguished by colors. Bar: 20 µm. B ) Relative signal intensity is shown in the graph.

    Techniques Used: Labeling, Mass Spectrometry, Incubation, Activity Assay

    Copper(I) treatment produces short gaps with phosphate groups at the 3′ end. A ) TdT was used to incorporate Alexa-dUTP at the 3′ end of the gaps. A strong signal is observed only after the pre-incubation of cells with exonuclease III or SAP. The model shows the situation after the action of SAP in the case of double-stranded DNA with several gaps. Although the phosphate groups are shown also at the 5′ end of the gaps, it is not clear whether they are present there. Therefore, the action of SAP is shown for 3′ phosphate groups exclusively. Bar: 20 µm. B ) DNA polymerase I, Klenow fragment and Klenow fragment Exo- were used to incorporate Alexa-dUTP at the gap sites produced by monovalent copper. Only DNA polymerase I produced a strong signal. When incubation with exonuclease III preceded the polymerase step, a strong signal was observed also in the case of both Klenow fragments. The model shows the action of DNA polymerase I at the sites of created gaps. Both 3′-5′ proofreading activity enabling hydroxyl group formation and 5′-3′ exonuclease activity (for the sake of simplicity, the excised nucleotides are not shown in the model) enabling nick translation are necessary. As no ligase activity was present, nicks at the ends of the labeled chains persisted (arrows in the model picture), although it is not apparent. Bar: 20 µm.
    Figure Legend Snippet: Copper(I) treatment produces short gaps with phosphate groups at the 3′ end. A ) TdT was used to incorporate Alexa-dUTP at the 3′ end of the gaps. A strong signal is observed only after the pre-incubation of cells with exonuclease III or SAP. The model shows the situation after the action of SAP in the case of double-stranded DNA with several gaps. Although the phosphate groups are shown also at the 5′ end of the gaps, it is not clear whether they are present there. Therefore, the action of SAP is shown for 3′ phosphate groups exclusively. Bar: 20 µm. B ) DNA polymerase I, Klenow fragment and Klenow fragment Exo- were used to incorporate Alexa-dUTP at the gap sites produced by monovalent copper. Only DNA polymerase I produced a strong signal. When incubation with exonuclease III preceded the polymerase step, a strong signal was observed also in the case of both Klenow fragments. The model shows the action of DNA polymerase I at the sites of created gaps. Both 3′-5′ proofreading activity enabling hydroxyl group formation and 5′-3′ exonuclease activity (for the sake of simplicity, the excised nucleotides are not shown in the model) enabling nick translation are necessary. As no ligase activity was present, nicks at the ends of the labeled chains persisted (arrows in the model picture), although it is not apparent. Bar: 20 µm.

    Techniques Used: Incubation, Produced, Activity Assay, Nick Translation, Labeling

    9) Product Images from "A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy"

    Article Title: A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy

    Journal: Cells

    doi: 10.3390/cells8121510

    Effect of fixation protocol on the signal intensity. The results of the detection of mycoplasmas’ DNA in samples with Lep cells accidentally infected with mycoplasma (RT-PCR-positive, MycoAlert-positive) using the developed approach and three fixation protocols is shown. The samples were fixed either with formaldehyde, with 70% ethanol, or in a solution of acetic acid and methanol (1:3). The enzymatic mixture contained biotin-dUTP. Biotin-dUTP was detected by indirect immunofluorescence (green). The overall DNA was labeled by DAPI (blue), mitochondria were labeled with the mitochondrial marker MTCO2 (red). The images were acquired for 16.56 ms (biotin-derived signal); 2.636 ms (DAPI) and 7.916 ms (MTCO2-derived signal). Scale bar = 10 µm.
    Figure Legend Snippet: Effect of fixation protocol on the signal intensity. The results of the detection of mycoplasmas’ DNA in samples with Lep cells accidentally infected with mycoplasma (RT-PCR-positive, MycoAlert-positive) using the developed approach and three fixation protocols is shown. The samples were fixed either with formaldehyde, with 70% ethanol, or in a solution of acetic acid and methanol (1:3). The enzymatic mixture contained biotin-dUTP. Biotin-dUTP was detected by indirect immunofluorescence (green). The overall DNA was labeled by DAPI (blue), mitochondria were labeled with the mitochondrial marker MTCO2 (red). The images were acquired for 16.56 ms (biotin-derived signal); 2.636 ms (DAPI) and 7.916 ms (MTCO2-derived signal). Scale bar = 10 µm.

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Labeling, Marker, Mass Spectrometry, Derivative Assay

    10) Product Images from "Potential implication of new torque teno mini viruses in parapneumonic empyema in children"

    Article Title: Potential implication of new torque teno mini viruses in parapneumonic empyema in children

    Journal: The European Respiratory Journal

    doi: 10.1183/09031936.00107212

    Replication of torque teno mini virus (TTMV) isolates TTMV-LY genomes after transfection produce more replicative TTMV in human embryonic kidney (HEK) 293T cells than in A549 cells as quantified by real-time qPCR. Histograms represent the mean± sd rate of replication of TTMV-LY in the cells transfected with the linearised full-length genomes. Replication of TTMV-LY was a) measured at day 3 post-transfection or b) monitored over a week following transfection. The curves in b) indicate the quantification of TTMV-LY1 in the culture medium of cells at days 0–7 post-transfection after DNaseI (Thermo Scientific, Illkirch, France) treatment and nucleic acid extraction. Results are expressed as the ratio of quantified TTMV-LY1 at day 1–7 post-transfection to quantified TTMV-LY1 at day 0. Data are shown only for TTMV-LY1, because the replication profile among the three viruses is similar.
    Figure Legend Snippet: Replication of torque teno mini virus (TTMV) isolates TTMV-LY genomes after transfection produce more replicative TTMV in human embryonic kidney (HEK) 293T cells than in A549 cells as quantified by real-time qPCR. Histograms represent the mean± sd rate of replication of TTMV-LY in the cells transfected with the linearised full-length genomes. Replication of TTMV-LY was a) measured at day 3 post-transfection or b) monitored over a week following transfection. The curves in b) indicate the quantification of TTMV-LY1 in the culture medium of cells at days 0–7 post-transfection after DNaseI (Thermo Scientific, Illkirch, France) treatment and nucleic acid extraction. Results are expressed as the ratio of quantified TTMV-LY1 at day 1–7 post-transfection to quantified TTMV-LY1 at day 0. Data are shown only for TTMV-LY1, because the replication profile among the three viruses is similar.

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction

    11) Product Images from "Cell cycle profiling by image and flow cytometry: The optimised protocol for the detection of replicational activity using 5-Bromo-2′-deoxyuridine, low concentration of hydrochloric acid and exonuclease III"

    Article Title: Cell cycle profiling by image and flow cytometry: The optimised protocol for the detection of replicational activity using 5-Bromo-2′-deoxyuridine, low concentration of hydrochloric acid and exonuclease III

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0175880

    Optimising the protocol for BrdU detection. (A) HeLa cells were incubated with BrdU for 30 min, fixed with formaldehyde and BrdU was detected in DNA using 40 mM HCl. BrdU was detected either by the B44 or Bu20a anti-BrdU antibody with exonuclease III. The effect of formaldehyde post-fixation on the signal is shown as well. The data are presented as the mean ± SD. ( B-E) A comparison of various HCl concentrations on BrdU signal in HeLa cells labelled for 30 min with BrdU and fixed either with formaldehyde (B, D) or ethanol (C, E). The incorporated BrdU was detected using either B44 (B, C) or Bu20a (D, E) antibody clone with exonuclease III. The data are presented as the mean ± SD. ( F, G) A comparison of five monoclonal anti-BrdU antibody clones and one polyclonal antibody is shown. The HeLa cells were labelled with BrdU for 30 min and fixed either with formaldehyde (F) or ethanol (G). The impact of the post-fixation step is shown as well. The data are presented as the mean ± SD. ( H) The effect of the length of the washing step on the BrdU-derived signal. The HeLa cells were labelled with BrdU for 30 min and fixed with formaldehyde. After incubation with the primary antibody, the cells were washed for 5 s (0 min) or 5 or 25 min in 1× PBS and then post-fixed with formaldehyde. The data are normalised to the % of the average signal in samples washed for 5 s in 1× PBS and then immediately post-fixed with formaldehyde. The data are presented as the mean ± SD.
    Figure Legend Snippet: Optimising the protocol for BrdU detection. (A) HeLa cells were incubated with BrdU for 30 min, fixed with formaldehyde and BrdU was detected in DNA using 40 mM HCl. BrdU was detected either by the B44 or Bu20a anti-BrdU antibody with exonuclease III. The effect of formaldehyde post-fixation on the signal is shown as well. The data are presented as the mean ± SD. ( B-E) A comparison of various HCl concentrations on BrdU signal in HeLa cells labelled for 30 min with BrdU and fixed either with formaldehyde (B, D) or ethanol (C, E). The incorporated BrdU was detected using either B44 (B, C) or Bu20a (D, E) antibody clone with exonuclease III. The data are presented as the mean ± SD. ( F, G) A comparison of five monoclonal anti-BrdU antibody clones and one polyclonal antibody is shown. The HeLa cells were labelled with BrdU for 30 min and fixed either with formaldehyde (F) or ethanol (G). The impact of the post-fixation step is shown as well. The data are presented as the mean ± SD. ( H) The effect of the length of the washing step on the BrdU-derived signal. The HeLa cells were labelled with BrdU for 30 min and fixed with formaldehyde. After incubation with the primary antibody, the cells were washed for 5 s (0 min) or 5 or 25 min in 1× PBS and then post-fixed with formaldehyde. The data are normalised to the % of the average signal in samples washed for 5 s in 1× PBS and then immediately post-fixed with formaldehyde. The data are presented as the mean ± SD.

    Techniques Used: Incubation, Clone Assay, Derivative Assay

    The effect of optimized procedure on the localisation of cellular proteins. HeLa cells were incubated with BrdU for 30 minutes and fixed with formaldehyde. BrdU was revealed using 20 mM HCl. The proteins SC35, mitochondrial protein MTCO2, histone H1.2 and coilin were concurrently detected with the incorporated BrdU. BrdU was detected using either chicken polyclonal antibody or B44 monoclonal antibody depending on the host producing the antibody for the particular cellular protein. The control cells were not labelled with BrdU and were not treated with HCl and exonuclease III. Proteins are in green, BrdU is in red and DAPI is in blue. Scale bar = 20 μm.
    Figure Legend Snippet: The effect of optimized procedure on the localisation of cellular proteins. HeLa cells were incubated with BrdU for 30 minutes and fixed with formaldehyde. BrdU was revealed using 20 mM HCl. The proteins SC35, mitochondrial protein MTCO2, histone H1.2 and coilin were concurrently detected with the incorporated BrdU. BrdU was detected using either chicken polyclonal antibody or B44 monoclonal antibody depending on the host producing the antibody for the particular cellular protein. The control cells were not labelled with BrdU and were not treated with HCl and exonuclease III. Proteins are in green, BrdU is in red and DAPI is in blue. Scale bar = 20 μm.

    Techniques Used: Incubation

    12) Product Images from "Copy-choice recombination during mitochondrial L-strand synthesis causes DNA deletions"

    Article Title: Copy-choice recombination during mitochondrial L-strand synthesis causes DNA deletions

    Journal: Nature Communications

    doi: 10.1038/s41467-019-08673-5

    mtDNA deletions identified in patients with mutations in POLγ. a Strand-displacement DNA replication is continuous on both strands and initiated from two separate origins. b mtDNA deletions (blue) and duplications (red) predicted using high-throughput sequencing of skeletal muscle DNA from three patients with pathogenic, compound heterozygous POLG variants as well as two healthy control patients. The orange bars in the outermost circle show breakpoint frequency at a given base position. c Frequencies of exact direct repeats overlapping or flanking each pair of breakpoints, considering the longest match for each deletion and pooling the three patients (red bars). Randomized breakpoints are shown for comparison (grey bars), with error bars indicating the standard deviation (100 randomizations). d Frequencies for the most commonly observed repeat patterns. e Analysis of 5′ vs. 3′ retention of imperfect repeats. A subset of deletions where the breakpoint positions could be safely determined to within 1 bp were analyzed for the presence of imperfect direct repeats at both sides of the deleted segment and were classified as either 5′ or 3′ based on the longest discovered repeat having at most 1 mismatch. Results are shown for unique deletions as well as the complete set of deletions, pooled from the three POLG patients. Results from 100 sets of randomly generated deletions, each being similar in size as the observed data, are included. Error bars indicate the standard deviation
    Figure Legend Snippet: mtDNA deletions identified in patients with mutations in POLγ. a Strand-displacement DNA replication is continuous on both strands and initiated from two separate origins. b mtDNA deletions (blue) and duplications (red) predicted using high-throughput sequencing of skeletal muscle DNA from three patients with pathogenic, compound heterozygous POLG variants as well as two healthy control patients. The orange bars in the outermost circle show breakpoint frequency at a given base position. c Frequencies of exact direct repeats overlapping or flanking each pair of breakpoints, considering the longest match for each deletion and pooling the three patients (red bars). Randomized breakpoints are shown for comparison (grey bars), with error bars indicating the standard deviation (100 randomizations). d Frequencies for the most commonly observed repeat patterns. e Analysis of 5′ vs. 3′ retention of imperfect repeats. A subset of deletions where the breakpoint positions could be safely determined to within 1 bp were analyzed for the presence of imperfect direct repeats at both sides of the deleted segment and were classified as either 5′ or 3′ based on the longest discovered repeat having at most 1 mismatch. Results are shown for unique deletions as well as the complete set of deletions, pooled from the three POLG patients. Results from 100 sets of randomly generated deletions, each being similar in size as the observed data, are included. Error bars indicate the standard deviation

    Techniques Used: Next-Generation Sequencing, Standard Deviation, Generated

    13) Product Images from "Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing"

    Article Title: Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

    Journal: mAbs

    doi: 10.4161/mabs.27435

    Figure 2. PCR checkpoint after clonal enrichment. ( A ) An agarose gel stained with ethidium bromide with inverted grayscale showing the PCR products of the clonal enrichment of group 2G3 and a negative control (mock enrichment). Two main products were amplified, one covering the whole VH region (VH-JH) and the other up to the CDRH3 (IGHV-CDRH3), of approximately 350 and 300 base pair, respectively. Three different amplification cycles were used (13X, 15X, and 18X) to determine the amplification threshold required for further processing. In the enriched sample, both products (IGHV-IGHJ and IGHV-CDRH3) are present, while they are slightly visible in 18X of the mock control. This difference demonstrates a selection for VH sequences containing the desired CDRH3. ( B ) PCR products of the ten enriched samples and their respective mock controls shown in separate agarose gels stained with ethidium bromide with an inverted grayscale. For each group, the enriched sample (left) and mock control (right) amplifications were performed with the indicated amplification cycles and annealing temperatures. The PCR products from the enriched samples shown here were used for cloning and sequencing.
    Figure Legend Snippet: Figure 2. PCR checkpoint after clonal enrichment. ( A ) An agarose gel stained with ethidium bromide with inverted grayscale showing the PCR products of the clonal enrichment of group 2G3 and a negative control (mock enrichment). Two main products were amplified, one covering the whole VH region (VH-JH) and the other up to the CDRH3 (IGHV-CDRH3), of approximately 350 and 300 base pair, respectively. Three different amplification cycles were used (13X, 15X, and 18X) to determine the amplification threshold required for further processing. In the enriched sample, both products (IGHV-IGHJ and IGHV-CDRH3) are present, while they are slightly visible in 18X of the mock control. This difference demonstrates a selection for VH sequences containing the desired CDRH3. ( B ) PCR products of the ten enriched samples and their respective mock controls shown in separate agarose gels stained with ethidium bromide with an inverted grayscale. For each group, the enriched sample (left) and mock control (right) amplifications were performed with the indicated amplification cycles and annealing temperatures. The PCR products from the enriched samples shown here were used for cloning and sequencing.

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Negative Control, Amplification, Selection, Clone Assay, Sequencing

    Figure 3. Enrichment assessment through single sequence analysis. ( A ) Representation of the concentrations of each of the eight clonal groups that were successfully recovered, before (gray) and after (yellow) the enrichment. Concentrations before enrichment were determined in silico by the analysis of the massive sequencing results; the number of sequences considered for 2G1–2G5 was 46 062, and 36 393 for 3G1–3G4. Concentrations after enrichment were determined through PCR amplification of the desired IGHV-CDRH3 fragment from cloned enrichment products; N indicates the total number of clones evaluated in each case. The circle representing the total (blue) is used as a reference of 100% of the area. ( B ) CDRH3 conservation after enrichment determined through Sanger sequencing. A few clones for each clonal group were sequenced (N) and compared with their respective target sequence, showing that most of the sequences obtained had no or little variation in the CDRH3. ( C ) HEL-binding ELISA for the three positive combinations (VH/VL) of the 32 tested. 2G3/HH10, 3G1/HH5 and 3G1/F10.6.6 showed HEL-binding capacity, corresponding to two VH of the eight tested. HH10/HH10 served as positive control, and no-DNA transfectant was used as negative control. The y-axis shows the ratio between the OD 490 from the HEL-binding ELISA and the OD 490 from the IgG detection ELISA of the same sample.
    Figure Legend Snippet: Figure 3. Enrichment assessment through single sequence analysis. ( A ) Representation of the concentrations of each of the eight clonal groups that were successfully recovered, before (gray) and after (yellow) the enrichment. Concentrations before enrichment were determined in silico by the analysis of the massive sequencing results; the number of sequences considered for 2G1–2G5 was 46 062, and 36 393 for 3G1–3G4. Concentrations after enrichment were determined through PCR amplification of the desired IGHV-CDRH3 fragment from cloned enrichment products; N indicates the total number of clones evaluated in each case. The circle representing the total (blue) is used as a reference of 100% of the area. ( B ) CDRH3 conservation after enrichment determined through Sanger sequencing. A few clones for each clonal group were sequenced (N) and compared with their respective target sequence, showing that most of the sequences obtained had no or little variation in the CDRH3. ( C ) HEL-binding ELISA for the three positive combinations (VH/VL) of the 32 tested. 2G3/HH10, 3G1/HH5 and 3G1/F10.6.6 showed HEL-binding capacity, corresponding to two VH of the eight tested. HH10/HH10 served as positive control, and no-DNA transfectant was used as negative control. The y-axis shows the ratio between the OD 490 from the HEL-binding ELISA and the OD 490 from the IgG detection ELISA of the same sample.

    Techniques Used: Sequencing, In Silico, Polymerase Chain Reaction, Amplification, Clone Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Positive Control, Transfection, Negative Control

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    Article Title: Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ
    Article Snippet: Enzymes used These enzymes and condition were used: Terminal deoxynucleotidyl transferase (TdT; 2 U/µl, 10 minutes, 37°C, Fermentas), buffer for TdT, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa Fluor® 555-aha-2′-deoxyuridine-5′-triphosphate (Alexa-dUTP); DNA polymerase I (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for DNA polymerase I, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Klenow fragment (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for the Klenow fragment, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Klenow fragment Exo- (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for the Klenow fragment Exo-, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Exonuclease III (1 U/µl, 30 minutes, RT, Fermentas), buffer for exonuclease III; Exonuclease λ (0.1 U/µl, 30 minutes, RT, Fermentas), buffer for exonuclease λ; Shrimp alkaline phosphomonoesterase (phosphatase; SAP; 1 U/µl, 20 minutes, 37°C, Fermentas), buffer for SAP.

    Plasmid Preparation:

    Article Title: DIRS retrotransposons amplify via linear, single-stranded cDNA intermediates
    Article Snippet: .. Nuclease treatment and detection of DIRS-1bsr extrachromosomal cDNA The extracted extrachromosomal cDNA sample (plasmid miniprep kit, as described before) was subjected to digestion with RNase A (Sigma-Aldrich), DNase I, exonuclease I, exonuclease III or S1 nuclease (Thermo Scientific). ..

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    Thermo Fisher exonuclease iii
    Features of extrachromosomal DIRS-1 bsr cDNA. Extrachromosomal cDNA samples were extracted from rrpC– strains transformed with DIRS-1 bsr ( A ) or DIRS-1 bsr* ( B ), and were cultivated in G418/BS10 medium that selects for strains with mobilized retrotransposon. The samples were treated separately with <t>RNase</t> A, DNase I, Exonuclease I (Exo I), Exonuclease <t>III</t> (Exo III) and S1 nuclease (S1). After digestion and heat inactivation of enzymes, the samples were analyzed by PCR with the primers #2212 and #2213 binding within the region of mbsrI cassette (binding position in Figure 2A ). Quantification of endogenous ( C and D ) and mbsrI -tagged versions ( E ) of DIRS-1 extrachromosomal cDNA after treatment with Exonuclease I and S1 nuclease, relative to the non-digested sample. Samples were extracted from rrpC– strains transformed with DIRS-1 bsr and DIRS-1 bsr* upon cultivation in G418/BS10 medium that selects for strains with mobilized retrotransposons. The cDNA abundance was monitored by qPCR using primers targeting positions qP1, qP2 and qBS (Figures 2A and 3A ) and is shown in logarithmic scale relative to the non-digested sample (set to 1). Each qPCR reaction was run in triplicate. Error bars : mean with S.D. Statistics: paired t test: P
    Exonuclease Iii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher exo iii
    Optimization of experimental conditions: (a) effect of the incubation time of <t>Exo</t> <t>III</t> digestion; (b) effect of Exo III concentration; (c) effect of signal DNA concentration; (d) effect of the incubation temperature; (e) effect of the signal DNA/capture DNA ratio; (f) effect of the pH value. Experimental conditions: 10 nmol/L oligo-1, 40 nmol/L oligo-2, 10 U Exo III, 50 mmol/L K + , and 160 nmol/L NMM. Error bars represent the standard deviation of three independent experiments.
    Exo Iii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher hiv gag qrt pcr dnase treated rna
    Viral gRNA levels analyzed by <t>qRT-PCR,</t> in the presence and absence of NSC. 293T cells were treated as in Fig. 2 . 24 h post treatment, supernatants and cells were harvested, virions purified from 500 μL supernatant and <t>RNA</t> extracted. a gRNA levels in cells, analyzed by qRT-PCR. Each sample was first normalized against β-actin levels and then normalized against the average wild-type gRNA levels. Data shown are the average of 14 –18 samples taken from four independent experiments. Error bars represent SD. b Ratio of gRNA levels in purified virions from equivalent volumes of supernatant to gRNA levels inside cells. Intracellular levels were first normalized against β-actin; these values were then used to divide the virion RNA levels for each sample. Data were then normalized against the wild-type average. Data are shown as three independent experiments, each containing 4–9 replicates. c Ratio of gRNA levels in purified virions from equivalent volumes of supernatant to cytoplasmic gRNA. Data are representative of two independent experiments. d Ability of virions produced in the presence or absence of NSC to transduce cells, measured by luciferase assay. Error bars represent the SD. * p
    Hiv Gag Qrt Pcr Dnase Treated Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Features of extrachromosomal DIRS-1 bsr cDNA. Extrachromosomal cDNA samples were extracted from rrpC– strains transformed with DIRS-1 bsr ( A ) or DIRS-1 bsr* ( B ), and were cultivated in G418/BS10 medium that selects for strains with mobilized retrotransposon. The samples were treated separately with RNase A, DNase I, Exonuclease I (Exo I), Exonuclease III (Exo III) and S1 nuclease (S1). After digestion and heat inactivation of enzymes, the samples were analyzed by PCR with the primers #2212 and #2213 binding within the region of mbsrI cassette (binding position in Figure 2A ). Quantification of endogenous ( C and D ) and mbsrI -tagged versions ( E ) of DIRS-1 extrachromosomal cDNA after treatment with Exonuclease I and S1 nuclease, relative to the non-digested sample. Samples were extracted from rrpC– strains transformed with DIRS-1 bsr and DIRS-1 bsr* upon cultivation in G418/BS10 medium that selects for strains with mobilized retrotransposons. The cDNA abundance was monitored by qPCR using primers targeting positions qP1, qP2 and qBS (Figures 2A and 3A ) and is shown in logarithmic scale relative to the non-digested sample (set to 1). Each qPCR reaction was run in triplicate. Error bars : mean with S.D. Statistics: paired t test: P

    Journal: Nucleic Acids Research

    Article Title: DIRS retrotransposons amplify via linear, single-stranded cDNA intermediates

    doi: 10.1093/nar/gkaa160

    Figure Lengend Snippet: Features of extrachromosomal DIRS-1 bsr cDNA. Extrachromosomal cDNA samples were extracted from rrpC– strains transformed with DIRS-1 bsr ( A ) or DIRS-1 bsr* ( B ), and were cultivated in G418/BS10 medium that selects for strains with mobilized retrotransposon. The samples were treated separately with RNase A, DNase I, Exonuclease I (Exo I), Exonuclease III (Exo III) and S1 nuclease (S1). After digestion and heat inactivation of enzymes, the samples were analyzed by PCR with the primers #2212 and #2213 binding within the region of mbsrI cassette (binding position in Figure 2A ). Quantification of endogenous ( C and D ) and mbsrI -tagged versions ( E ) of DIRS-1 extrachromosomal cDNA after treatment with Exonuclease I and S1 nuclease, relative to the non-digested sample. Samples were extracted from rrpC– strains transformed with DIRS-1 bsr and DIRS-1 bsr* upon cultivation in G418/BS10 medium that selects for strains with mobilized retrotransposons. The cDNA abundance was monitored by qPCR using primers targeting positions qP1, qP2 and qBS (Figures 2A and 3A ) and is shown in logarithmic scale relative to the non-digested sample (set to 1). Each qPCR reaction was run in triplicate. Error bars : mean with S.D. Statistics: paired t test: P

    Article Snippet: Nuclease treatment and detection of DIRS-1bsr extrachromosomal cDNA The extracted extrachromosomal cDNA sample (plasmid miniprep kit, as described before) was subjected to digestion with RNase A (Sigma-Aldrich), DNase I, exonuclease I, exonuclease III or S1 nuclease (Thermo Scientific).

    Techniques: Transformation Assay, Polymerase Chain Reaction, Binding Assay, Real-time Polymerase Chain Reaction

    Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells. NP4000 membrane, which can detect particles with a diameter between 1.0 and 6.0 μm, was used for quantitation of L-EVs, while NP200 membrane, which can detect particles with a diameter between 60 and 400 nm, was used for quantitation of S-EVs. (b) Protein lysates from L-EVs and S-EVs purified by iodixanol density gradient (at 1.10 and 1.15 g/ml) were blotted with LO markers HSPA5 and CK18, and with Exo marker CD81. (c) Total DNA was quantified by Qubit Fluorometer in L-EVs and S-EVs isolated from PC3 and U87 cell lines. The plot shows the DNA ratio between L-EVs and S-EVs. (d) Double stranded (ds)DNA was quantified by High Sensitivity (HS) dsDNA Qubit Assay in L-EVs and S-EVs isolated from 1 ml of plasma from patients with mCRPC ( n = 40) and cancer-free individuals ( n = 6). (e) Quantification of both protein and DNA content in L-EVs and S-EVs isolated from conditioned media of 12.6 × 10 7 PC3 cells. (f) Single stranded (ss) and dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Exonuclease III, were quantified by Qubit. (g) Chip-based capillary electrophoresis (Bioanalyzer) showing the presence of dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Endonuclease III. L-EVs contain abundant DNA with a large peak around 10 kbp. Conversely, the amount of DNA in S-EVs is negligible. (h) ss- and dsDNA in PC3-derived L-EVs and S-EVs were quantified by Qubit after treatment with nucleases (DNase I and Exonuclease III) with or without addition of a detergent (Triton X-100) prior to nuclease treatment. (i) Chip-based capillary electrophoresis (Bioanalyzer) showing that only miniscule amounts of dsDNA could be detected after EV lysis using a detergent prior to treatment with nucleases.

    Journal: Journal of Extracellular Vesicles

    Article Title: Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma

    doi: 10.1080/20013078.2018.1505403

    Figure Lengend Snippet: Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells. NP4000 membrane, which can detect particles with a diameter between 1.0 and 6.0 μm, was used for quantitation of L-EVs, while NP200 membrane, which can detect particles with a diameter between 60 and 400 nm, was used for quantitation of S-EVs. (b) Protein lysates from L-EVs and S-EVs purified by iodixanol density gradient (at 1.10 and 1.15 g/ml) were blotted with LO markers HSPA5 and CK18, and with Exo marker CD81. (c) Total DNA was quantified by Qubit Fluorometer in L-EVs and S-EVs isolated from PC3 and U87 cell lines. The plot shows the DNA ratio between L-EVs and S-EVs. (d) Double stranded (ds)DNA was quantified by High Sensitivity (HS) dsDNA Qubit Assay in L-EVs and S-EVs isolated from 1 ml of plasma from patients with mCRPC ( n = 40) and cancer-free individuals ( n = 6). (e) Quantification of both protein and DNA content in L-EVs and S-EVs isolated from conditioned media of 12.6 × 10 7 PC3 cells. (f) Single stranded (ss) and dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Exonuclease III, were quantified by Qubit. (g) Chip-based capillary electrophoresis (Bioanalyzer) showing the presence of dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Endonuclease III. L-EVs contain abundant DNA with a large peak around 10 kbp. Conversely, the amount of DNA in S-EVs is negligible. (h) ss- and dsDNA in PC3-derived L-EVs and S-EVs were quantified by Qubit after treatment with nucleases (DNase I and Exonuclease III) with or without addition of a detergent (Triton X-100) prior to nuclease treatment. (i) Chip-based capillary electrophoresis (Bioanalyzer) showing that only miniscule amounts of dsDNA could be detected after EV lysis using a detergent prior to treatment with nucleases.

    Article Snippet: DNA extraction and characterization L- and S-EVs were treated with rDNase I (2 U/μl, DNA-Free Kit, Ambion) and Exonuclease III (200 U/μl, Thermofisher).

    Techniques: Tunable Resistive Pulse Sensing, Derivative Assay, Quantitation Assay, Purification, Marker, Isolation, HS DSDNA Qubit Assay, Chromatin Immunoprecipitation, Electrophoresis, Lysis

    Optimization of experimental conditions: (a) effect of the incubation time of Exo III digestion; (b) effect of Exo III concentration; (c) effect of signal DNA concentration; (d) effect of the incubation temperature; (e) effect of the signal DNA/capture DNA ratio; (f) effect of the pH value. Experimental conditions: 10 nmol/L oligo-1, 40 nmol/L oligo-2, 10 U Exo III, 50 mmol/L K + , and 160 nmol/L NMM. Error bars represent the standard deviation of three independent experiments.

    Journal: Journal of Analytical Methods in Chemistry

    Article Title: A Sensitive Fluorescence Biosensor for Silver Ions (Ag+) Detection Based on C-Ag+-C Structure and Exonuclease III-Assisted Dual-Recycling Amplification

    doi: 10.1155/2019/3712032

    Figure Lengend Snippet: Optimization of experimental conditions: (a) effect of the incubation time of Exo III digestion; (b) effect of Exo III concentration; (c) effect of signal DNA concentration; (d) effect of the incubation temperature; (e) effect of the signal DNA/capture DNA ratio; (f) effect of the pH value. Experimental conditions: 10 nmol/L oligo-1, 40 nmol/L oligo-2, 10 U Exo III, 50 mmol/L K + , and 160 nmol/L NMM. Error bars represent the standard deviation of three independent experiments.

    Article Snippet: Exo III was purchased from the Thermo Fisher Scientific Inc. (USA).

    Techniques: Incubation, Concentration Assay, Standard Deviation

    Fluorescence spectra (a) and calibration plot (b) for Ag + . Experimental conditions: 10 nmol/L oligo-1, 40 nmol/L oligo-2, 10 U Exo III, 50 mmol/L K + , 160 nmol/L NMM, and 50 min incubation time. Error bars represent the standard deviation of three independent experiments.

    Journal: Journal of Analytical Methods in Chemistry

    Article Title: A Sensitive Fluorescence Biosensor for Silver Ions (Ag+) Detection Based on C-Ag+-C Structure and Exonuclease III-Assisted Dual-Recycling Amplification

    doi: 10.1155/2019/3712032

    Figure Lengend Snippet: Fluorescence spectra (a) and calibration plot (b) for Ag + . Experimental conditions: 10 nmol/L oligo-1, 40 nmol/L oligo-2, 10 U Exo III, 50 mmol/L K + , 160 nmol/L NMM, and 50 min incubation time. Error bars represent the standard deviation of three independent experiments.

    Article Snippet: Exo III was purchased from the Thermo Fisher Scientific Inc. (USA).

    Techniques: Fluorescence, Incubation, Standard Deviation

    Selectivity of the biosensor. Experimental conditions: 10 nmol/L oligo-1, 40 nmol/L oligo-2, 10 U Exo III, 50 mmol/L K + , 160 nmol/L NMM, and 50 min incubation time. The concentration of Ag + was 1.5 nmol/L, and the concentrations of other metal ions were 15 nmol/L. Error bars represent the standard deviation of three independent experiments.

    Journal: Journal of Analytical Methods in Chemistry

    Article Title: A Sensitive Fluorescence Biosensor for Silver Ions (Ag+) Detection Based on C-Ag+-C Structure and Exonuclease III-Assisted Dual-Recycling Amplification

    doi: 10.1155/2019/3712032

    Figure Lengend Snippet: Selectivity of the biosensor. Experimental conditions: 10 nmol/L oligo-1, 40 nmol/L oligo-2, 10 U Exo III, 50 mmol/L K + , 160 nmol/L NMM, and 50 min incubation time. The concentration of Ag + was 1.5 nmol/L, and the concentrations of other metal ions were 15 nmol/L. Error bars represent the standard deviation of three independent experiments.

    Article Snippet: Exo III was purchased from the Thermo Fisher Scientific Inc. (USA).

    Techniques: Incubation, Concentration Assay, Standard Deviation

    Fluorescence emission spectra of different solutions: (a) 10 nmol/L oligo-1 + 1500 pmol/L Ag + ; (b) 40 nmol/L oligo-2 + 1500 pmol/L Ag + ; (c) 10 nmol/L oligo-1 + 40 nmol/L oligo-2; (d) 10 nmol/L oligo-1 + 40 nmol/L oligo-2 + 10 U Exo III; (e) 10 nmol/L oligo-1 + 40 nmol/L oligo-2 + 10 U Exo III + 1500 pmol/L Ag + . Experimental conditions: 50 mmol/L K + and 160 nmol/L NMM.

    Journal: Journal of Analytical Methods in Chemistry

    Article Title: A Sensitive Fluorescence Biosensor for Silver Ions (Ag+) Detection Based on C-Ag+-C Structure and Exonuclease III-Assisted Dual-Recycling Amplification

    doi: 10.1155/2019/3712032

    Figure Lengend Snippet: Fluorescence emission spectra of different solutions: (a) 10 nmol/L oligo-1 + 1500 pmol/L Ag + ; (b) 40 nmol/L oligo-2 + 1500 pmol/L Ag + ; (c) 10 nmol/L oligo-1 + 40 nmol/L oligo-2; (d) 10 nmol/L oligo-1 + 40 nmol/L oligo-2 + 10 U Exo III; (e) 10 nmol/L oligo-1 + 40 nmol/L oligo-2 + 10 U Exo III + 1500 pmol/L Ag + . Experimental conditions: 50 mmol/L K + and 160 nmol/L NMM.

    Article Snippet: Exo III was purchased from the Thermo Fisher Scientific Inc. (USA).

    Techniques: Fluorescence

    Viral gRNA levels analyzed by qRT-PCR, in the presence and absence of NSC. 293T cells were treated as in Fig. 2 . 24 h post treatment, supernatants and cells were harvested, virions purified from 500 μL supernatant and RNA extracted. a gRNA levels in cells, analyzed by qRT-PCR. Each sample was first normalized against β-actin levels and then normalized against the average wild-type gRNA levels. Data shown are the average of 14 –18 samples taken from four independent experiments. Error bars represent SD. b Ratio of gRNA levels in purified virions from equivalent volumes of supernatant to gRNA levels inside cells. Intracellular levels were first normalized against β-actin; these values were then used to divide the virion RNA levels for each sample. Data were then normalized against the wild-type average. Data are shown as three independent experiments, each containing 4–9 replicates. c Ratio of gRNA levels in purified virions from equivalent volumes of supernatant to cytoplasmic gRNA. Data are representative of two independent experiments. d Ability of virions produced in the presence or absence of NSC to transduce cells, measured by luciferase assay. Error bars represent the SD. * p

    Journal: Retrovirology

    Article Title: An RNA-binding compound that stabilizes the HIV-1 gRNA packaging signal structure and specifically blocks HIV-1 RNA encapsidation

    doi: 10.1186/s12977-018-0407-4

    Figure Lengend Snippet: Viral gRNA levels analyzed by qRT-PCR, in the presence and absence of NSC. 293T cells were treated as in Fig. 2 . 24 h post treatment, supernatants and cells were harvested, virions purified from 500 μL supernatant and RNA extracted. a gRNA levels in cells, analyzed by qRT-PCR. Each sample was first normalized against β-actin levels and then normalized against the average wild-type gRNA levels. Data shown are the average of 14 –18 samples taken from four independent experiments. Error bars represent SD. b Ratio of gRNA levels in purified virions from equivalent volumes of supernatant to gRNA levels inside cells. Intracellular levels were first normalized against β-actin; these values were then used to divide the virion RNA levels for each sample. Data were then normalized against the wild-type average. Data are shown as three independent experiments, each containing 4–9 replicates. c Ratio of gRNA levels in purified virions from equivalent volumes of supernatant to cytoplasmic gRNA. Data are representative of two independent experiments. d Ability of virions produced in the presence or absence of NSC to transduce cells, measured by luciferase assay. Error bars represent the SD. * p

    Article Snippet: HIV gag qRT-PCR DNase-treated RNA was reverse transcribed with the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions with RNase inhibitor added to the reaction, or with the addition of RNasin® Ribonuclease Inhibitor (Promega).

    Techniques: Quantitative RT-PCR, Purification, Produced, Transduction, Luciferase