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  • 99
    New England Biolabs streptavidin coated magnetic beads
    General scheme applied for identifying peanut immature pod-specific genes (tracer mRNA (1)) after a single round subtraction . B biotin, S <t>streptavidin,</t> M magnetic bead.
    Streptavidin Coated Magnetic Beads, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 360 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 360 article reviews
    Price from $9.99 to $1999.99
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    99
    New England Biolabs streptavidin magnetic beads
    Validating biotin-14-dCTP incorporation by a bind and boil method. The <t>biotin–streptavidin</t> dissociation constant (kD) is on the order of 4 × 10 −14 mol/L. However, boiling the biotin-steptavidin complexes in the presence of SDS releases these noncovalent complexes. To validate the incorporation of biotin-14-dCTP during the PCR, we assayed the supernatant of our PCR “bait” solution after mixing with Dynal M-280 beads. If biotin-14-dCTP did not incorporate during the PCR, we would expect the supernatant to contain PCR-amplified DNA. To assay the supernatant, we performed a buffer exchange using AmpureXP beads, eluted in water, and evaluated the supernatant of PCR on the Agilent BioAnalyzer 2100 High Sensitivity DNA chip (red dotted line). The Dynal M-280 beads were boiled in 0.1% SDS, and this supernatant was assayed for PCR products (dotted blue line). A second boil treatment was performed, and this supernatant was assayed for the presence of PCR products (dotted green line). FU, fluorescent units; nt, nucleotides.
    Streptavidin Magnetic Beads, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 789 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin magnetic beads/product/New England Biolabs
    Average 99 stars, based on 789 article reviews
    Price from $9.99 to $1999.99
    streptavidin magnetic beads - by Bioz Stars, 2020-09
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    99
    New England Biolabs magnetic beads
    Validating biotin-14-dCTP incorporation by a bind and boil method. The <t>biotin–streptavidin</t> dissociation constant (kD) is on the order of 4 × 10 −14 mol/L. However, boiling the biotin-steptavidin complexes in the presence of SDS releases these noncovalent complexes. To validate the incorporation of biotin-14-dCTP during the PCR, we assayed the supernatant of our PCR “bait” solution after mixing with Dynal M-280 beads. If biotin-14-dCTP did not incorporate during the PCR, we would expect the supernatant to contain PCR-amplified DNA. To assay the supernatant, we performed a buffer exchange using AmpureXP beads, eluted in water, and evaluated the supernatant of PCR on the Agilent BioAnalyzer 2100 High Sensitivity DNA chip (red dotted line). The Dynal M-280 beads were boiled in 0.1% SDS, and this supernatant was assayed for PCR products (dotted blue line). A second boil treatment was performed, and this supernatant was assayed for the presence of PCR products (dotted green line). FU, fluorescent units; nt, nucleotides.
    Magnetic Beads, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 265 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/magnetic beads/product/New England Biolabs
    Average 99 stars, based on 265 article reviews
    Price from $9.99 to $1999.99
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    99
    Promega streptavidin coated magnetic beads
    Validating biotin-14-dCTP incorporation by a bind and boil method. The <t>biotin–streptavidin</t> dissociation constant (kD) is on the order of 4 × 10 −14 mol/L. However, boiling the biotin-steptavidin complexes in the presence of SDS releases these noncovalent complexes. To validate the incorporation of biotin-14-dCTP during the PCR, we assayed the supernatant of our PCR “bait” solution after mixing with Dynal M-280 beads. If biotin-14-dCTP did not incorporate during the PCR, we would expect the supernatant to contain PCR-amplified DNA. To assay the supernatant, we performed a buffer exchange using AmpureXP beads, eluted in water, and evaluated the supernatant of PCR on the Agilent BioAnalyzer 2100 High Sensitivity DNA chip (red dotted line). The Dynal M-280 beads were boiled in 0.1% SDS, and this supernatant was assayed for PCR products (dotted blue line). A second boil treatment was performed, and this supernatant was assayed for the presence of PCR products (dotted green line). FU, fluorescent units; nt, nucleotides.
    Streptavidin Coated Magnetic Beads, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1088 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin coated magnetic beads/product/Promega
    Average 99 stars, based on 1088 article reviews
    Price from $9.99 to $1999.99
    streptavidin coated magnetic beads - by Bioz Stars, 2020-09
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    88
    Thermo Fisher streptavidin coated magnetic microbeads
    Validating biotin-14-dCTP incorporation by a bind and boil method. The <t>biotin–streptavidin</t> dissociation constant (kD) is on the order of 4 × 10 −14 mol/L. However, boiling the biotin-steptavidin complexes in the presence of SDS releases these noncovalent complexes. To validate the incorporation of biotin-14-dCTP during the PCR, we assayed the supernatant of our PCR “bait” solution after mixing with Dynal M-280 beads. If biotin-14-dCTP did not incorporate during the PCR, we would expect the supernatant to contain PCR-amplified DNA. To assay the supernatant, we performed a buffer exchange using AmpureXP beads, eluted in water, and evaluated the supernatant of PCR on the Agilent BioAnalyzer 2100 High Sensitivity DNA chip (red dotted line). The Dynal M-280 beads were boiled in 0.1% SDS, and this supernatant was assayed for PCR products (dotted blue line). A second boil treatment was performed, and this supernatant was assayed for the presence of PCR products (dotted green line). FU, fluorescent units; nt, nucleotides.
    Streptavidin Coated Magnetic Microbeads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs nuclease bal 31
    Validating biotin-14-dCTP incorporation by a bind and boil method. The <t>biotin–streptavidin</t> dissociation constant (kD) is on the order of 4 × 10 −14 mol/L. However, boiling the biotin-steptavidin complexes in the presence of SDS releases these noncovalent complexes. To validate the incorporation of biotin-14-dCTP during the PCR, we assayed the supernatant of our PCR “bait” solution after mixing with Dynal M-280 beads. If biotin-14-dCTP did not incorporate during the PCR, we would expect the supernatant to contain PCR-amplified DNA. To assay the supernatant, we performed a buffer exchange using AmpureXP beads, eluted in water, and evaluated the supernatant of PCR on the Agilent BioAnalyzer 2100 High Sensitivity DNA chip (red dotted line). The Dynal M-280 beads were boiled in 0.1% SDS, and this supernatant was assayed for PCR products (dotted blue line). A second boil treatment was performed, and this supernatant was assayed for the presence of PCR products (dotted green line). FU, fluorescent units; nt, nucleotides.
    Nuclease Bal 31, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    General scheme applied for identifying peanut immature pod-specific genes (tracer mRNA (1)) after a single round subtraction . B biotin, S streptavidin, M magnetic bead.

    Journal: Biological Procedures Online

    Article Title: A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

    doi: 10.1007/s12575-009-9022-z

    Figure Lengend Snippet: General scheme applied for identifying peanut immature pod-specific genes (tracer mRNA (1)) after a single round subtraction . B biotin, S streptavidin, M magnetic bead.

    Article Snippet: Streptavidin-coated magnetic beads (1.1 mg/ml) were applied again to purify synthesized double-stranded cDNAs with three times washes.

    Techniques:

    Selection of streptavidin-binding DeNAno. ( A ) Schematic of selection process. ( B ) Staining of streptavidin selection rounds 1–5. Probe-only, library and positive control (biotinylated library) are also shown. ( C ) Staining of four selected streptavidin clones on streptavidin beads and BSA-coated beads. Negative control clone (G10neg) and biotinylated positive control clone (G10bio) also shown.

    Journal: Nucleic Acids Research

    Article Title: Selection of DNA nanoparticles with preferential binding to aggregated protein target

    doi: 10.1093/nar/gkw136

    Figure Lengend Snippet: Selection of streptavidin-binding DeNAno. ( A ) Schematic of selection process. ( B ) Staining of streptavidin selection rounds 1–5. Probe-only, library and positive control (biotinylated library) are also shown. ( C ) Staining of four selected streptavidin clones on streptavidin beads and BSA-coated beads. Negative control clone (G10neg) and biotinylated positive control clone (G10bio) also shown.

    Article Snippet: Beads Streptavidin-coated magnetic beads (NEB) were used for selections/staining for streptavidin-specific DeNAno with no modifications.

    Techniques: Selection, Binding Assay, Staining, Positive Control, Clone Assay, Negative Control

    Competitive titration and competitive release of streptavidin-binding DeNAno with biotin/biotin derivatives or streptavidin. ( A ) Schematic of competitive titration using biotin/biotin derivatives. ( B ) Free biotin (top), desthiobiotin (middle) and 2-iminobiotin (bottom) competition titrations were done by pre-incubating streptavidin beads with one of the biotin/biotin derivatives (or buffer for the baseline), then adding DeNAno particles. ( C ) Schematic of competitive release using biotin/biotin derivatives. ( D ) Biotin (top), desthiobiotin (middle) and 2-iminobiotin (bottom) competitive release assays were done by staining streptavidin beads with DeNAno particles, then adding biotin/biotin derivative (or buffer for baseline). ( E ) Schematic of streptavidin competitive titration. ( F ) Free streptavidin competition titration of SA-D7 and SA-D8 clones and G10bio positive control. Fluorescently-labeled DeNAno particles were pre-incubated with varying concentrations of free streptavidin, then streptavidin beads were added.

    Journal: Nucleic Acids Research

    Article Title: Selection of DNA nanoparticles with preferential binding to aggregated protein target

    doi: 10.1093/nar/gkw136

    Figure Lengend Snippet: Competitive titration and competitive release of streptavidin-binding DeNAno with biotin/biotin derivatives or streptavidin. ( A ) Schematic of competitive titration using biotin/biotin derivatives. ( B ) Free biotin (top), desthiobiotin (middle) and 2-iminobiotin (bottom) competition titrations were done by pre-incubating streptavidin beads with one of the biotin/biotin derivatives (or buffer for the baseline), then adding DeNAno particles. ( C ) Schematic of competitive release using biotin/biotin derivatives. ( D ) Biotin (top), desthiobiotin (middle) and 2-iminobiotin (bottom) competitive release assays were done by staining streptavidin beads with DeNAno particles, then adding biotin/biotin derivative (or buffer for baseline). ( E ) Schematic of streptavidin competitive titration. ( F ) Free streptavidin competition titration of SA-D7 and SA-D8 clones and G10bio positive control. Fluorescently-labeled DeNAno particles were pre-incubated with varying concentrations of free streptavidin, then streptavidin beads were added.

    Article Snippet: Beads Streptavidin-coated magnetic beads (NEB) were used for selections/staining for streptavidin-specific DeNAno with no modifications.

    Techniques: Titration, Binding Assay, Staining, Clone Assay, Positive Control, Labeling, Incubation

    Dissociation of streptavidin-binding DeNAno over time. Streptavidin-coated magnetic beads were stained with DeNAno particles. The stained beads were then incubated in 10 ml buffer for 35 days. Aliquots were taken every week of the total sample (supernatant plus beads) and supernatant only (beads were removed by magnet). PCR was done on all samples/timepoints and percentage release is graphed (DeNAno in supernatant/DeNAno in total * 100%).

    Journal: Nucleic Acids Research

    Article Title: Selection of DNA nanoparticles with preferential binding to aggregated protein target

    doi: 10.1093/nar/gkw136

    Figure Lengend Snippet: Dissociation of streptavidin-binding DeNAno over time. Streptavidin-coated magnetic beads were stained with DeNAno particles. The stained beads were then incubated in 10 ml buffer for 35 days. Aliquots were taken every week of the total sample (supernatant plus beads) and supernatant only (beads were removed by magnet). PCR was done on all samples/timepoints and percentage release is graphed (DeNAno in supernatant/DeNAno in total * 100%).

    Article Snippet: Beads Streptavidin-coated magnetic beads (NEB) were used for selections/staining for streptavidin-specific DeNAno with no modifications.

    Techniques: Binding Assay, Magnetic Beads, Staining, Incubation, Polymerase Chain Reaction

    Imaging of DeNAno and staining different size DeNAno. ( A ) Atomic force micrograph (AFM) of dried DeNAno SA-D8 on poly-L-lysine-coated mica. Scale = 400 nm. ( B ) SA-D8 DeNAno roughly 75 nm in diameter as observed by transmission electron microscopy (TEM) using negative staining. Scale = 100 nm. ( C ) TEM of small SA-D8 DeNAno roughly 58 nm in diameter. Scale = 100 nm. ( D ) Binding of streptavidin DeNAno of different sizes made by alteration of dNTP concentration. DeNAno particles were made with 3 nmol dNTPs for 30 m at 30°C (the standard conditions), or 93.8 pmol dNTPs for 30 m at 30°C. A control DeNAno from a different library was also made for both of these conditions and used as an internal control in the staining and subsequent PCR. The ratio of the bound particles (streptavidin DeNAno:control DeNano) to total particles (streptavidin DeNAno:control DeNAno) is graphed.

    Journal: Nucleic Acids Research

    Article Title: Selection of DNA nanoparticles with preferential binding to aggregated protein target

    doi: 10.1093/nar/gkw136

    Figure Lengend Snippet: Imaging of DeNAno and staining different size DeNAno. ( A ) Atomic force micrograph (AFM) of dried DeNAno SA-D8 on poly-L-lysine-coated mica. Scale = 400 nm. ( B ) SA-D8 DeNAno roughly 75 nm in diameter as observed by transmission electron microscopy (TEM) using negative staining. Scale = 100 nm. ( C ) TEM of small SA-D8 DeNAno roughly 58 nm in diameter. Scale = 100 nm. ( D ) Binding of streptavidin DeNAno of different sizes made by alteration of dNTP concentration. DeNAno particles were made with 3 nmol dNTPs for 30 m at 30°C (the standard conditions), or 93.8 pmol dNTPs for 30 m at 30°C. A control DeNAno from a different library was also made for both of these conditions and used as an internal control in the staining and subsequent PCR. The ratio of the bound particles (streptavidin DeNAno:control DeNano) to total particles (streptavidin DeNAno:control DeNAno) is graphed.

    Article Snippet: Beads Streptavidin-coated magnetic beads (NEB) were used for selections/staining for streptavidin-specific DeNAno with no modifications.

    Techniques: Imaging, Staining, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Negative Staining, Binding Assay, Concentration Assay, Polymerase Chain Reaction

    In vitro dephosphorylation assays and generation of RPTP chimeras. ( A ) The indicated PTPRK and PTPRM domains were assayed for phosphatase activity using the pNPP colorimetric assay. Control wells contained pNPP only. Protein amounts used are shown. ( B ) Pervanadate-treated MCF10A lysates were incubated with predetermined amounts of the indicated domains to give equal phosphatase-activity, prior to phosphotyrosine immunoprecipitation and immunoblot analysis. ( C ) Recombinant proteins consisting of combinations of PTPRK and PTPRM D1 and D2 domains were expressed in and using Ni-NTA affinity resin. Purified proteins were then subjected to size exclusion chromatography. ( D ) Recombinant His- and Avi-tagged PTPRK and PTPRM chimeric domains were purified from E. coli cultured in biotin-supplemented media, incubated ±streptavidin and subjected to SDS-PAGE and Coomassie staining, to determine the extent of biotinylation. Arrows indicate the purified domains and the respective streptavidin-induced mobility shift. ( E ) The indicated recombinant PTPRK and PTPRM chimeric domains were incubated were assayed for phosphatase activity using the pNPP colorimetric assay. Control wells contained pNPP. Protein amounts used are shown.

    Journal: eLife

    Article Title: The homophilic receptor PTPRK selectively dephosphorylates multiple junctional regulators to promote cell–cell adhesion

    doi: 10.7554/eLife.44597

    Figure Lengend Snippet: In vitro dephosphorylation assays and generation of RPTP chimeras. ( A ) The indicated PTPRK and PTPRM domains were assayed for phosphatase activity using the pNPP colorimetric assay. Control wells contained pNPP only. Protein amounts used are shown. ( B ) Pervanadate-treated MCF10A lysates were incubated with predetermined amounts of the indicated domains to give equal phosphatase-activity, prior to phosphotyrosine immunoprecipitation and immunoblot analysis. ( C ) Recombinant proteins consisting of combinations of PTPRK and PTPRM D1 and D2 domains were expressed in and using Ni-NTA affinity resin. Purified proteins were then subjected to size exclusion chromatography. ( D ) Recombinant His- and Avi-tagged PTPRK and PTPRM chimeric domains were purified from E. coli cultured in biotin-supplemented media, incubated ±streptavidin and subjected to SDS-PAGE and Coomassie staining, to determine the extent of biotinylation. Arrows indicate the purified domains and the respective streptavidin-induced mobility shift. ( E ) The indicated recombinant PTPRK and PTPRM chimeric domains were incubated were assayed for phosphatase activity using the pNPP colorimetric assay. Control wells contained pNPP. Protein amounts used are shown.

    Article Snippet: Recombinant protein pull downs 25–50 μg (tandem or single domain, respectively) of biotinylated His.TEV.Avi.PTPx domains were conjugated to 167 μl of pre-washed streptavidin-coated magnetic beads suspension (4 mg/ml; New England Biolabs) in 500 μl of ice-cold size exclusion buffer (50 mM HEPES pH 7.5 (50 mM Tris pH 7.4 for SH2 domains), 150 mM NaCl, 5% (v/v) glycerol, 5 mM DTT) at 4°C for 1–2 hr on a rotator.

    Techniques: In Vitro, De-Phosphorylation Assay, Activity Assay, Colorimetric Assay, Incubation, Immunoprecipitation, Recombinant, Purification, Size-exclusion Chromatography, Cell Culture, SDS Page, Staining, Mobility Shift

    Purification of biotinylated recombinant PTPRK domains. ( A ) His- and Avi-tagged PTPRK domains were expressed in E. coli cultured in biotin-supplemented media and purified using Nickel-NTA beads, followed by size exclusion chromatography (SEC). DA = D1057A mutant. CS = C1089S mutant. ( B ) SEC-purified proteins bound to streptavidin resin were eluted and resolved by SDS-PAGE followed by Coomassie staining. In; input, B; beads. ( C ) The phosphatase activity of indicated amounts of purified proteins was assessed using the Biomol green assay with two tyrosine phosphorylated peptides as substrates and was quantified at 620 nm. ( D ) Recombinant proteins bound to streptavidin resin were used in pull down assays from pervanadate treated Hs27 fibroblast lysates. After extensive washing, bound proteins were eluted in sample buffer and analyzed by immunoblot.

    Journal: eLife

    Article Title: The homophilic receptor PTPRK selectively dephosphorylates multiple junctional regulators to promote cell–cell adhesion

    doi: 10.7554/eLife.44597

    Figure Lengend Snippet: Purification of biotinylated recombinant PTPRK domains. ( A ) His- and Avi-tagged PTPRK domains were expressed in E. coli cultured in biotin-supplemented media and purified using Nickel-NTA beads, followed by size exclusion chromatography (SEC). DA = D1057A mutant. CS = C1089S mutant. ( B ) SEC-purified proteins bound to streptavidin resin were eluted and resolved by SDS-PAGE followed by Coomassie staining. In; input, B; beads. ( C ) The phosphatase activity of indicated amounts of purified proteins was assessed using the Biomol green assay with two tyrosine phosphorylated peptides as substrates and was quantified at 620 nm. ( D ) Recombinant proteins bound to streptavidin resin were used in pull down assays from pervanadate treated Hs27 fibroblast lysates. After extensive washing, bound proteins were eluted in sample buffer and analyzed by immunoblot.

    Article Snippet: Recombinant protein pull downs 25–50 μg (tandem or single domain, respectively) of biotinylated His.TEV.Avi.PTPx domains were conjugated to 167 μl of pre-washed streptavidin-coated magnetic beads suspension (4 mg/ml; New England Biolabs) in 500 μl of ice-cold size exclusion buffer (50 mM HEPES pH 7.5 (50 mM Tris pH 7.4 for SH2 domains), 150 mM NaCl, 5% (v/v) glycerol, 5 mM DTT) at 4°C for 1–2 hr on a rotator.

    Techniques: Purification, Recombinant, Cell Culture, Size-exclusion Chromatography, Mutagenesis, SDS Page, Staining, Activity Assay

    The PTPRK-dependent tyrosine phosphoproteome. ( A ) After SEC, proteins were incubated with or without streptavidin and subjected to SDS PAGE followed by Coomassie staining to determine the extent of biotinylation. Arrows indicate the purified domains and the respective streptavidin-induced mobility shift. ( B ) Volcano plot of tyrosine phosphosites detected in PTPRK KO and wildtype MCF10As. Phosphosites > 50% enriched in (p

    Journal: eLife

    Article Title: The homophilic receptor PTPRK selectively dephosphorylates multiple junctional regulators to promote cell–cell adhesion

    doi: 10.7554/eLife.44597

    Figure Lengend Snippet: The PTPRK-dependent tyrosine phosphoproteome. ( A ) After SEC, proteins were incubated with or without streptavidin and subjected to SDS PAGE followed by Coomassie staining to determine the extent of biotinylation. Arrows indicate the purified domains and the respective streptavidin-induced mobility shift. ( B ) Volcano plot of tyrosine phosphosites detected in PTPRK KO and wildtype MCF10As. Phosphosites > 50% enriched in (p

    Article Snippet: Recombinant protein pull downs 25–50 μg (tandem or single domain, respectively) of biotinylated His.TEV.Avi.PTPx domains were conjugated to 167 μl of pre-washed streptavidin-coated magnetic beads suspension (4 mg/ml; New England Biolabs) in 500 μl of ice-cold size exclusion buffer (50 mM HEPES pH 7.5 (50 mM Tris pH 7.4 for SH2 domains), 150 mM NaCl, 5% (v/v) glycerol, 5 mM DTT) at 4°C for 1–2 hr on a rotator.

    Techniques: Size-exclusion Chromatography, Incubation, SDS Page, Staining, Purification, Mobility Shift

    Validating biotin-14-dCTP incorporation by a bind and boil method. The biotin–streptavidin dissociation constant (kD) is on the order of 4 × 10 −14 mol/L. However, boiling the biotin-steptavidin complexes in the presence of SDS releases these noncovalent complexes. To validate the incorporation of biotin-14-dCTP during the PCR, we assayed the supernatant of our PCR “bait” solution after mixing with Dynal M-280 beads. If biotin-14-dCTP did not incorporate during the PCR, we would expect the supernatant to contain PCR-amplified DNA. To assay the supernatant, we performed a buffer exchange using AmpureXP beads, eluted in water, and evaluated the supernatant of PCR on the Agilent BioAnalyzer 2100 High Sensitivity DNA chip (red dotted line). The Dynal M-280 beads were boiled in 0.1% SDS, and this supernatant was assayed for PCR products (dotted blue line). A second boil treatment was performed, and this supernatant was assayed for the presence of PCR products (dotted green line). FU, fluorescent units; nt, nucleotides.

    Journal: The Journal of Molecular Diagnostics : JMD

    Article Title: Hybrid Capture and Next-Generation Sequencing Identify Viral Integration Sites from Formalin-Fixed, Paraffin-Embedded Tissue

    doi: 10.1016/j.jmoldx.2011.01.006

    Figure Lengend Snippet: Validating biotin-14-dCTP incorporation by a bind and boil method. The biotin–streptavidin dissociation constant (kD) is on the order of 4 × 10 −14 mol/L. However, boiling the biotin-steptavidin complexes in the presence of SDS releases these noncovalent complexes. To validate the incorporation of biotin-14-dCTP during the PCR, we assayed the supernatant of our PCR “bait” solution after mixing with Dynal M-280 beads. If biotin-14-dCTP did not incorporate during the PCR, we would expect the supernatant to contain PCR-amplified DNA. To assay the supernatant, we performed a buffer exchange using AmpureXP beads, eluted in water, and evaluated the supernatant of PCR on the Agilent BioAnalyzer 2100 High Sensitivity DNA chip (red dotted line). The Dynal M-280 beads were boiled in 0.1% SDS, and this supernatant was assayed for PCR products (dotted blue line). A second boil treatment was performed, and this supernatant was assayed for the presence of PCR products (dotted green line). FU, fluorescent units; nt, nucleotides.

    Article Snippet: Briefly, the capture probes were assayed for biotin incorporation by binding 500 ng of biotinylated PCR products to 200 μg of streptavidin-coated paramagnetic beads (cat. #S1420S; New England Biolabs, Worcester, MA).

    Techniques: Polymerase Chain Reaction, Amplification, Buffer Exchange, Chromatin Immunoprecipitation

    Pictorial representation of Washington University Capture. Washington University Capture (WUCap) enables solution-phase hybridization between double-stranded DNA PCR “bait” and whole-genome shotgun libraries. The solution-phase method we have developed for hybrid capture is robust and involves only a few basic steps. The bait used for targeting is dictated by primer-specific amplification of genomic targets generated during the PCR. Subsequently, the amplicons are used as a template in a second PCR incorporating biotin-14-dCTP. Genomic DNA is prepared from each of the samples to be sequenced, sheared to an average fragment size of 300 bp, enzymatically repaired to blunt the ends, and ligated to Illumina adapter sequences (at both ends). Five hundred nanograms of genomic DNA library is denatured, combined with 100 ng of the biotinylated “bait,” and hybridized for 48 hours. Mixing this hybridization reaction with streptavidin-coated superparamagnetic beads allows binding of biotinylated bait–target hybrids and selective removal from solution by applying a magnet field. The remaining supernatant is removed, and the beads are washed, removing nonspecific DNA. The enriched target sequences are released from the bead-bound bait sequences by denaturation (0.125 N NaOH), neutralized, amplified in the PCR to generate double-stranded Illumina libraries, and then sequenced.

    Journal: The Journal of Molecular Diagnostics : JMD

    Article Title: Hybrid Capture and Next-Generation Sequencing Identify Viral Integration Sites from Formalin-Fixed, Paraffin-Embedded Tissue

    doi: 10.1016/j.jmoldx.2011.01.006

    Figure Lengend Snippet: Pictorial representation of Washington University Capture. Washington University Capture (WUCap) enables solution-phase hybridization between double-stranded DNA PCR “bait” and whole-genome shotgun libraries. The solution-phase method we have developed for hybrid capture is robust and involves only a few basic steps. The bait used for targeting is dictated by primer-specific amplification of genomic targets generated during the PCR. Subsequently, the amplicons are used as a template in a second PCR incorporating biotin-14-dCTP. Genomic DNA is prepared from each of the samples to be sequenced, sheared to an average fragment size of 300 bp, enzymatically repaired to blunt the ends, and ligated to Illumina adapter sequences (at both ends). Five hundred nanograms of genomic DNA library is denatured, combined with 100 ng of the biotinylated “bait,” and hybridized for 48 hours. Mixing this hybridization reaction with streptavidin-coated superparamagnetic beads allows binding of biotinylated bait–target hybrids and selective removal from solution by applying a magnet field. The remaining supernatant is removed, and the beads are washed, removing nonspecific DNA. The enriched target sequences are released from the bead-bound bait sequences by denaturation (0.125 N NaOH), neutralized, amplified in the PCR to generate double-stranded Illumina libraries, and then sequenced.

    Article Snippet: Briefly, the capture probes were assayed for biotin incorporation by binding 500 ng of biotinylated PCR products to 200 μg of streptavidin-coated paramagnetic beads (cat. #S1420S; New England Biolabs, Worcester, MA).

    Techniques: Hybridization, Polymerase Chain Reaction, Amplification, Generated, Binding Assay