magnetic hydrophilic streptavidin beads  (New England Biolabs)


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    Hydrophilic Streptavidin Magnetic Beads
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    Hydrophilic Streptavidin Magnetic Beads 5 ml
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    New England Biolabs magnetic hydrophilic streptavidin beads
    Hydrophilic Streptavidin Magnetic Beads
    Hydrophilic Streptavidin Magnetic Beads 5 ml
    https://www.bioz.com/result/magnetic hydrophilic streptavidin beads/product/New England Biolabs
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    magnetic hydrophilic streptavidin beads - by Bioz Stars, 2020-04
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    1) Product Images from "An efficient and sensitive method for preparing cDNA libraries from scarce biological samples"

    Article Title: An efficient and sensitive method for preparing cDNA libraries from scarce biological samples

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku637

    Overview of 3-day generation of cDNA libraries. ( A ) On the first day, total RNA is ligated to a 3′ adapter and cDNA is generated by reverse transcription by tandem reactions in a single tube, RNA is degraded and cDNAs are isolated by ethanol precipitation. ( B ) On the second day, cDNAs are circularized, size selected by gel fractionation and eluted overnight in the presence of streptavidin beads. ( C ) PCR is done on bead-bound purified cDNAs to generate templates ready for high-throughput sequencing.
    Figure Legend Snippet: Overview of 3-day generation of cDNA libraries. ( A ) On the first day, total RNA is ligated to a 3′ adapter and cDNA is generated by reverse transcription by tandem reactions in a single tube, RNA is degraded and cDNAs are isolated by ethanol precipitation. ( B ) On the second day, cDNAs are circularized, size selected by gel fractionation and eluted overnight in the presence of streptavidin beads. ( C ) PCR is done on bead-bound purified cDNAs to generate templates ready for high-throughput sequencing.

    Techniques Used: Generated, Isolation, Ethanol Precipitation, Fractionation, Polymerase Chain Reaction, Purification, Next-Generation Sequencing

    Detailed LQ cloning method. ( A ) A pre-adenylated (rApp) 3′-terminal dideoxy-C (ddC) blocked adapter (gray) is annealed to a ssDNA reverse transcription (RT) oligo (black) in a 1:1 molar ratio. The annealed adapter is ligated to 3′-hydroxyl-containing RNA (orange) using T4 RNA Ligase 2 (truncated K227Q) without ATP. Each RT oligo contains a 5′ Guanine (G) followed by a 4 or 6 nucleotide randomer (N X ), a 3–6 nucleotide barcode (BAR) and 3 internal deoxyUridine (dU) nucleotides. The adapter::RT oligo hybrid is in excess over RNA, resulting in free adapter::primer material present in the completed reaction. ( B ) Reverse transcription of ligated RNA is carried out in the same tube as the ligation reaction generating ‘+ insert’ and ‘no insert’ cDNA products (red and black line) using dGTP, dTTP, dATP, dCTP as well as biotinylated dATP and dCTP (yellow ‘B’-containing circles). The RNA template is degraded (dashed orange line) by base hydrolysis and cDNA is ethanol precipitated with ammonium acetate to facilitate maximum removal of free adapter and unincorporated nucleotides ( C ). Ethanol precipitated cDNAs are circularized ( D ) and resolved on a 10% denaturing polyacrylamide gel. ‘+ insert’ circularized cDNAs are isolated by excising and eluting them from the gel overnight in the presence of magnetic streptavidin beads ( E ). Bead-bound ‘+ insert’ cDNAs serve as templates in the first round of PCR. Amplification is done using a mix containing uracil-N-deglycosylase (UNG) to remove dU nucleotides, thereby generating a linear template through strand scission, and with primers complimentary to the 3′ adapter (blue) and 5′ end of the RT oligo (tan) ( F ). First round PCR products are resolved on an 8% native polyacrylamide gel, the 60–70 nucleotide products are excised and a portion is used as the template for second round PCR. Second round PCR products are generated using primers complimentary to the 3′ adapter (dark blue) and 5′ end of the RT oligo (brown) that contain the full Illumina or Ion Torrent adapter sequences (dark blue and brown) ( G ).
    Figure Legend Snippet: Detailed LQ cloning method. ( A ) A pre-adenylated (rApp) 3′-terminal dideoxy-C (ddC) blocked adapter (gray) is annealed to a ssDNA reverse transcription (RT) oligo (black) in a 1:1 molar ratio. The annealed adapter is ligated to 3′-hydroxyl-containing RNA (orange) using T4 RNA Ligase 2 (truncated K227Q) without ATP. Each RT oligo contains a 5′ Guanine (G) followed by a 4 or 6 nucleotide randomer (N X ), a 3–6 nucleotide barcode (BAR) and 3 internal deoxyUridine (dU) nucleotides. The adapter::RT oligo hybrid is in excess over RNA, resulting in free adapter::primer material present in the completed reaction. ( B ) Reverse transcription of ligated RNA is carried out in the same tube as the ligation reaction generating ‘+ insert’ and ‘no insert’ cDNA products (red and black line) using dGTP, dTTP, dATP, dCTP as well as biotinylated dATP and dCTP (yellow ‘B’-containing circles). The RNA template is degraded (dashed orange line) by base hydrolysis and cDNA is ethanol precipitated with ammonium acetate to facilitate maximum removal of free adapter and unincorporated nucleotides ( C ). Ethanol precipitated cDNAs are circularized ( D ) and resolved on a 10% denaturing polyacrylamide gel. ‘+ insert’ circularized cDNAs are isolated by excising and eluting them from the gel overnight in the presence of magnetic streptavidin beads ( E ). Bead-bound ‘+ insert’ cDNAs serve as templates in the first round of PCR. Amplification is done using a mix containing uracil-N-deglycosylase (UNG) to remove dU nucleotides, thereby generating a linear template through strand scission, and with primers complimentary to the 3′ adapter (blue) and 5′ end of the RT oligo (tan) ( F ). First round PCR products are resolved on an 8% native polyacrylamide gel, the 60–70 nucleotide products are excised and a portion is used as the template for second round PCR. Second round PCR products are generated using primers complimentary to the 3′ adapter (dark blue) and 5′ end of the RT oligo (brown) that contain the full Illumina or Ion Torrent adapter sequences (dark blue and brown) ( G ).

    Techniques Used: Clone Assay, Ligation, Isolation, Polymerase Chain Reaction, Amplification, Generated

    Related Articles

    Luciferase:

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: In order to measure the recovery of triphosphate transcripts, 1 ng of in vitro synthesized Gluc (Gaussia Luciferase) transcripts were mixed with the E. coli RNA for the capping and following reactions. .. The capped RNA was enriched using hydrophilic streptavidin magnetic beads (New England Biolabs).

    Synthesized:

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: In order to measure the recovery of triphosphate transcripts, 1 ng of in vitro synthesized Gluc (Gaussia Luciferase) transcripts were mixed with the E. coli RNA for the capping and following reactions. .. The capped RNA was enriched using hydrophilic streptavidin magnetic beads (New England Biolabs).

    Incubation:

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: The capped RNA was enriched using hydrophilic streptavidin magnetic beads (New England Biolabs). .. A volume of 40 μl of capped and tailed RNA was incubated with the beads at room temperature for 30 min on a rotator.

    Article Title: A Short Tandem Repeat-Enriched RNA Assembles a Nuclear Compartment to Control Alternative Splicing and Promote Cell Survival
    Article Snippet: The RNA-cDNA duplexes were precipitated by 5 μL of 10% SDS, 5 μL of 5 M NaCl, 75 μL of 1 M NaOAc (pH 6.1), 0.5 μL of glycogen (20 μg/μl) and 720 μL of 100% ethanol and incubated for 1 hour at −20°C. .. To capture these biotinylated duplexes, we used hydrophilic streptavidin magnetic beads (New England Biolabs, cat#S1421S) prepared in the following manner.

    Article Title: Lysine-specific demethylase 2A enhances binding of various nuclear factors to CpG-rich genomic DNAs by action of its CXXC-PHD domain
    Article Snippet: .. Four pmol bait was bound to hydrophilic streptavidin magnetic beads (NEB, S1421S), and incubated with 200 µg nuclear extracts in 50 µl up to 30 min at room temperature. ..

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: .. The purified cDNA/RNA duplex samples were incubated with 10 units TdT and 2 mM dGTP at 37 °C for 30 min. For the SMRT-Cappable-seq RNA, the reaction was purified using 30 μl hydrophilic streptavidin magnetic beads (New England Biolabs) as mentioned above but without Biotin buffer elution. ..

    Article Title: Detection of sub-microscopic blood levels of Plasmodium falciparum using Tandem Oligonucleotide Repeat Cascade Amplification (TORCA) assay with an attomolar detection limit
    Article Snippet: Attachment of the recognition probe to magnetic beads Hydrophilic Streptavidin Magnetic Bead suspension (200 µL of 4 mg/mL stock suspension, New England Biolabs) was settled and washed 2 times with 200 µL 1X PBS. .. Finally, the pre-washed beads were incubated with 200 µL of 1 µM solution of a recognition probe oligonucleotide (bio-polyC-ST-SP, bio-polyC-CL-SP, and bio-polyC-HN-SP, Table ) or mixture of all three recognition probes (1 µM each) in 1X PBS.

    Article Title: Aβ-dependent reduction of NCAM2-mediated synaptic adhesion contributes to synapse loss in Alzheimer's disease
    Article Snippet: Peptides (250 ng ml−1 final concentration) were mixed with lysates of hippocampal tissue or synaptosomes (20 μg of total protein in 1 ml of TBS) and incubated for 1 h at room temperature. .. Non-cleaved peptides and biotin-containing fragments of cleaved peptide were removed by incubating lysates with hydrophilic streptavidin magnetic beads (New England BioLabs) for 1 h at room temperature.

    Article Title: The Neuroprotective Marine Compound Psammaplysene A Binds the RNA-Binding Protein HNRNPK
    Article Snippet: .. Biotin-tagged lysates were incubated with hydrophilic streptavidin magnetic beads (New England Biolabs, Ipswich, MA, USA) at 4 °C for 1 h with rotation. .. Beads were washed three times with high stringency wash buffer (PBS with 1 M KCl and 500 mM Urea) to decrease pulldown of indirect binding partners.

    Mass Spectrometry:

    Article Title: Lysine-specific demethylase 2A enhances binding of various nuclear factors to CpG-rich genomic DNAs by action of its CXXC-PHD domain
    Article Snippet: Four pmol bait was bound to hydrophilic streptavidin magnetic beads (NEB, S1421S), and incubated with 200 µg nuclear extracts in 50 µl up to 30 min at room temperature. .. For mass spectrometry, the IGFBPL1-171 bait (4 pmol) was incubated with 500 µg nuclear extracts in 72 µl for 10 min.

    Modification:

    Article Title: A Short Tandem Repeat-Enriched RNA Assembles a Nuclear Compartment to Control Alternative Splicing and Promote Cell Survival
    Article Snippet: At this point, cDNA-RNA duplexes corresponding to the 5′ ends of capped RNAs should be covalently modified by biotin groups in the periodate-oxidized/biocytin hydrazide-treated sample (but not in the negative control). .. To capture these biotinylated duplexes, we used hydrophilic streptavidin magnetic beads (New England Biolabs, cat#S1421S) prepared in the following manner.

    Western Blot:

    Article Title: An efficient and sensitive method for preparing cDNA libraries from scarce biological samples
    Article Snippet: .. 5.0 μl magnetic hydrophilic streptavidin beads (New England Biolabs) were washed three times with 50 μl buffer WB (0.5M NaCl, 20 mM Tris-HCl pH 7.5, 1.0 mM EDTA), resuspended in 5.0 μl TE + 0.3M NaCl and added to each sample. .. After overnight elution, the bead-containing supernatant was transferred to a clean, low-retention 1.7 ml eppendorf tube, tubes were magnetized on a magnetic rack (Life Technologies), supernatants carefully removed, beads washed three times with 1.0 ml buffer WB (magnetizing and carefully removing supernatant between each wash step) and resuspended in 10.0 μl RNase free H2 O.

    Ligation:

    Article Title: Chromatin run-on and sequencing maps the transcriptional regulatory landscape of glioblastoma multiforme
    Article Snippet: Nascent RNA was purified by binding streptavidin beads (NEB # S1421S) and washed as described . .. RNA was removed from beads by Trizol and followed by the 3’ adapter ligation (NEB # M0204L).

    Protease Inhibitor:

    Article Title: Aβ-dependent reduction of NCAM2-mediated synaptic adhesion contributes to synapse loss in Alzheimer's disease
    Article Snippet: Peptide cleavage assay Hippocampal tissue or synaptosomes containing 1 mg of total protein were lysed with lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na4 P2 O7 , 1 mM NaF, 1% (v/v) Triton X-100, protease inhibitor cocktail (set III, Merck)) for 30 min at room temperature. .. Non-cleaved peptides and biotin-containing fragments of cleaved peptide were removed by incubating lysates with hydrophilic streptavidin magnetic beads (New England BioLabs) for 1 h at room temperature.

    Imaging:

    Article Title: The Neuroprotective Marine Compound Psammaplysene A Binds the RNA-Binding Protein HNRNPK
    Article Snippet: TAMRA-tagged lysates were run on a 4–15% gradient gel and then visualized by in-gel laser scanning with the Typhoon 9400 imaging system (GE Life Sciences, Marlborough, MA, USA). .. Biotin-tagged lysates were incubated with hydrophilic streptavidin magnetic beads (New England Biolabs, Ipswich, MA, USA) at 4 °C for 1 h with rotation.

    Sequencing:

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Paragraph title: cDNA synthesis for SMRT Sequencing ... The purified cDNA/RNA duplex samples were incubated with 10 units TdT and 2 mM dGTP at 37 °C for 30 min. For the SMRT-Cappable-seq RNA, the reaction was purified using 30 μl hydrophilic streptavidin magnetic beads (New England Biolabs) as mentioned above but without Biotin buffer elution.

    Article Title: Chromatin run-on and sequencing maps the transcriptional regulatory landscape of glioblastoma multiforme
    Article Snippet: Paragraph title: Chromatin Run-On and sequencing (ChRO-seq) library preparation. ... Nascent RNA was purified by binding streptavidin beads (NEB # S1421S) and washed as described .

    Binding Assay:

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: The capped RNA was enriched using hydrophilic streptavidin magnetic beads (New England Biolabs). .. A volume of 40 μl of beads were prepared by washing with Washing Buffer and suspended in 40 μl Binding Buffer (see below for composition).

    Article Title: Global repositioning of transcription start sites in a plant-fermenting bacterium
    Article Snippet: .. Streptavidin magnetic beads (NEB S1421S) were pre-washed twice with low-salt buffer (10 mM Tris, 50 mM NaCl, 1 mM EDTA), twice with binding buffer (10 mM Tris, 500 mM NaCl, 1 mM EDTA) and resuspended at 4 mg ml−1 beads in binding buffer. .. Capped RNA fragments were bound to streptavidin beads for 20 min at room temperature and magnetically separated from other RNA by washing twice with binding buffer and twice with low-salt buffer to elute non-bound RNA.

    Article Title: Chromatin run-on and sequencing maps the transcriptional regulatory landscape of glioblastoma multiforme
    Article Snippet: .. Nascent RNA was purified by binding streptavidin beads (NEB # S1421S) and washed as described . .. RNA was removed from beads by Trizol and followed by the 3’ adapter ligation (NEB # M0204L).

    Article Title: The Neuroprotective Marine Compound Psammaplysene A Binds the RNA-Binding Protein HNRNPK
    Article Snippet: Biotin-tagged lysates were incubated with hydrophilic streptavidin magnetic beads (New England Biolabs, Ipswich, MA, USA) at 4 °C for 1 h with rotation. .. Beads were washed three times with high stringency wash buffer (PBS with 1 M KCl and 500 mM Urea) to decrease pulldown of indirect binding partners.

    Molecular Weight:

    Article Title: An efficient and sensitive method for preparing cDNA libraries from scarce biological samples
    Article Snippet: Gel fractionation, elution and isolation of biotinylated cDNA Circularized cDNAs and circular single-stranded DNA molecular weight markers were fractionated on separate lanes of a 10% polyacrylamide/7M urea gel. .. 5.0 μl magnetic hydrophilic streptavidin beads (New England Biolabs) were washed three times with 50 μl buffer WB (0.5M NaCl, 20 mM Tris-HCl pH 7.5, 1.0 mM EDTA), resuspended in 5.0 μl TE + 0.3M NaCl and added to each sample.

    Cleavage Assay:

    Article Title: Aβ-dependent reduction of NCAM2-mediated synaptic adhesion contributes to synapse loss in Alzheimer's disease
    Article Snippet: Paragraph title: Peptide cleavage assay ... Non-cleaved peptides and biotin-containing fragments of cleaved peptide were removed by incubating lysates with hydrophilic streptavidin magnetic beads (New England BioLabs) for 1 h at room temperature.

    Fluorescence:

    Article Title: Aβ-dependent reduction of NCAM2-mediated synaptic adhesion contributes to synapse loss in Alzheimer's disease
    Article Snippet: Non-cleaved peptides and biotin-containing fragments of cleaved peptide were removed by incubating lysates with hydrophilic streptavidin magnetic beads (New England BioLabs) for 1 h at room temperature. .. Fluorescence of FITC groups attached to fragments of cleaved peptides remaining in the solution was measured using POLARstar Omega plate reader (BMG Labtech).

    Magnetic Beads:

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: .. The capped RNA was enriched using hydrophilic streptavidin magnetic beads (New England Biolabs). .. A volume of 40 μl of beads were prepared by washing with Washing Buffer and suspended in 40 μl Binding Buffer (see below for composition).

    Article Title: Global repositioning of transcription start sites in a plant-fermenting bacterium
    Article Snippet: .. Streptavidin magnetic beads (NEB S1421S) were pre-washed twice with low-salt buffer (10 mM Tris, 50 mM NaCl, 1 mM EDTA), twice with binding buffer (10 mM Tris, 500 mM NaCl, 1 mM EDTA) and resuspended at 4 mg ml−1 beads in binding buffer. .. Capped RNA fragments were bound to streptavidin beads for 20 min at room temperature and magnetically separated from other RNA by washing twice with binding buffer and twice with low-salt buffer to elute non-bound RNA.

    Article Title: A Short Tandem Repeat-Enriched RNA Assembles a Nuclear Compartment to Control Alternative Splicing and Promote Cell Survival
    Article Snippet: .. To capture these biotinylated duplexes, we used hydrophilic streptavidin magnetic beads (New England Biolabs, cat#S1421S) prepared in the following manner. ..

    Article Title: Lysine-specific demethylase 2A enhances binding of various nuclear factors to CpG-rich genomic DNAs by action of its CXXC-PHD domain
    Article Snippet: .. Four pmol bait was bound to hydrophilic streptavidin magnetic beads (NEB, S1421S), and incubated with 200 µg nuclear extracts in 50 µl up to 30 min at room temperature. ..

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: .. The purified cDNA/RNA duplex samples were incubated with 10 units TdT and 2 mM dGTP at 37 °C for 30 min. For the SMRT-Cappable-seq RNA, the reaction was purified using 30 μl hydrophilic streptavidin magnetic beads (New England Biolabs) as mentioned above but without Biotin buffer elution. ..

    Article Title: Detection of sub-microscopic blood levels of Plasmodium falciparum using Tandem Oligonucleotide Repeat Cascade Amplification (TORCA) assay with an attomolar detection limit
    Article Snippet: .. Attachment of the recognition probe to magnetic beads Hydrophilic Streptavidin Magnetic Bead suspension (200 µL of 4 mg/mL stock suspension, New England Biolabs) was settled and washed 2 times with 200 µL 1X PBS. ..

    Article Title: Aβ-dependent reduction of NCAM2-mediated synaptic adhesion contributes to synapse loss in Alzheimer's disease
    Article Snippet: .. Non-cleaved peptides and biotin-containing fragments of cleaved peptide were removed by incubating lysates with hydrophilic streptavidin magnetic beads (New England BioLabs) for 1 h at room temperature. .. Fluorescence of FITC groups attached to fragments of cleaved peptides remaining in the solution was measured using POLARstar Omega plate reader (BMG Labtech).

    Article Title: The Neuroprotective Marine Compound Psammaplysene A Binds the RNA-Binding Protein HNRNPK
    Article Snippet: .. Biotin-tagged lysates were incubated with hydrophilic streptavidin magnetic beads (New England Biolabs, Ipswich, MA, USA) at 4 °C for 1 h with rotation. .. Beads were washed three times with high stringency wash buffer (PBS with 1 M KCl and 500 mM Urea) to decrease pulldown of indirect binding partners.

    Isolation:

    Article Title: An efficient and sensitive method for preparing cDNA libraries from scarce biological samples
    Article Snippet: Paragraph title: Gel fractionation, elution and isolation of biotinylated cDNA ... 5.0 μl magnetic hydrophilic streptavidin beads (New England Biolabs) were washed three times with 50 μl buffer WB (0.5M NaCl, 20 mM Tris-HCl pH 7.5, 1.0 mM EDTA), resuspended in 5.0 μl TE + 0.3M NaCl and added to each sample.

    Purification:

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: The capped and tailed RNA was purified using AMPure beads and eluted in 50 μl low TE buffer. .. The capped RNA was enriched using hydrophilic streptavidin magnetic beads (New England Biolabs).

    Article Title: Global repositioning of transcription start sites in a plant-fermenting bacterium
    Article Snippet: RNA was fragmented for 30 s at 94 °C using NEBNext Magnesium-based RNA fragmentation buffer (NEB E6101) and purified by Zymo Concentrator-5 (total RNA capture) into 100 μl water. .. Streptavidin magnetic beads (NEB S1421S) were pre-washed twice with low-salt buffer (10 mM Tris, 50 mM NaCl, 1 mM EDTA), twice with binding buffer (10 mM Tris, 500 mM NaCl, 1 mM EDTA) and resuspended at 4 mg ml−1 beads in binding buffer.

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: .. The purified cDNA/RNA duplex samples were incubated with 10 units TdT and 2 mM dGTP at 37 °C for 30 min. For the SMRT-Cappable-seq RNA, the reaction was purified using 30 μl hydrophilic streptavidin magnetic beads (New England Biolabs) as mentioned above but without Biotin buffer elution. ..

    Article Title: Chromatin run-on and sequencing maps the transcriptional regulatory landscape of glioblastoma multiforme
    Article Snippet: .. Nascent RNA was purified by binding streptavidin beads (NEB # S1421S) and washed as described . .. RNA was removed from beads by Trizol and followed by the 3’ adapter ligation (NEB # M0204L).

    Article Title: The Neuroprotective Marine Compound Psammaplysene A Binds the RNA-Binding Protein HNRNPK
    Article Snippet: Biotin-tagged lysates were incubated with hydrophilic streptavidin magnetic beads (New England Biolabs, Ipswich, MA, USA) at 4 °C for 1 h with rotation. .. Purification of PA Targets with PA-FG Beads: HEK293 lysates were incubated with 0X, 1X, and 5X PA-FG beads for 1 h at 4 °C.

    Polymerase Chain Reaction:

    Article Title: Lysine-specific demethylase 2A enhances binding of various nuclear factors to CpG-rich genomic DNAs by action of its CXXC-PHD domain
    Article Snippet: DNA-protein interaction Bait used in this study was prepared by PCR including a 5′ end biotinylated forward primer and a backward primer. .. Four pmol bait was bound to hydrophilic streptavidin magnetic beads (NEB, S1421S), and incubated with 200 µg nuclear extracts in 50 µl up to 30 min at room temperature.

    Lysis:

    Article Title: Aβ-dependent reduction of NCAM2-mediated synaptic adhesion contributes to synapse loss in Alzheimer's disease
    Article Snippet: Peptide cleavage assay Hippocampal tissue or synaptosomes containing 1 mg of total protein were lysed with lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na4 P2 O7 , 1 mM NaF, 1% (v/v) Triton X-100, protease inhibitor cocktail (set III, Merck)) for 30 min at room temperature. .. Non-cleaved peptides and biotin-containing fragments of cleaved peptide were removed by incubating lysates with hydrophilic streptavidin magnetic beads (New England BioLabs) for 1 h at room temperature.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: A Short Tandem Repeat-Enriched RNA Assembles a Nuclear Compartment to Control Alternative Splicing and Promote Cell Survival
    Article Snippet: .. To capture these biotinylated duplexes, we used hydrophilic streptavidin magnetic beads (New England Biolabs, catS1421S) prepared in the following manner. ..

    Negative Control:

    Article Title: A Short Tandem Repeat-Enriched RNA Assembles a Nuclear Compartment to Control Alternative Splicing and Promote Cell Survival
    Article Snippet: At this point, cDNA-RNA duplexes corresponding to the 5′ ends of capped RNAs should be covalently modified by biotin groups in the periodate-oxidized/biocytin hydrazide-treated sample (but not in the negative control). .. To capture these biotinylated duplexes, we used hydrophilic streptavidin magnetic beads (New England Biolabs, cat#S1421S) prepared in the following manner.

    In Vitro:

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Next, a polyA tail was added in vitro by incubating the capped RNA in 50 μl reaction volume with 20 units E. coli Poly(A) Polymerase (New England Biolabs) and 1 mM ATP for 15 min at 37 °C. .. The capped RNA was enriched using hydrophilic streptavidin magnetic beads (New England Biolabs).

    Concentration Assay:

    Article Title: Aβ-dependent reduction of NCAM2-mediated synaptic adhesion contributes to synapse loss in Alzheimer's disease
    Article Snippet: Peptides (250 ng ml−1 final concentration) were mixed with lysates of hippocampal tissue or synaptosomes (20 μg of total protein in 1 ml of TBS) and incubated for 1 h at room temperature. .. Non-cleaved peptides and biotin-containing fragments of cleaved peptide were removed by incubating lysates with hydrophilic streptavidin magnetic beads (New England BioLabs) for 1 h at room temperature.

    Fractionation:

    Article Title: An efficient and sensitive method for preparing cDNA libraries from scarce biological samples
    Article Snippet: Paragraph title: Gel fractionation, elution and isolation of biotinylated cDNA ... 5.0 μl magnetic hydrophilic streptavidin beads (New England Biolabs) were washed three times with 50 μl buffer WB (0.5M NaCl, 20 mM Tris-HCl pH 7.5, 1.0 mM EDTA), resuspended in 5.0 μl TE + 0.3M NaCl and added to each sample.

    Staining:

    Article Title: An efficient and sensitive method for preparing cDNA libraries from scarce biological samples
    Article Snippet: The gel was stained with SYBR Gold (Life Technologies) according to manufacturer's instructions, visualized on a blue light transilluminator and circularized cDNAs migrating in the range of 80 –100 nt were excised. .. 5.0 μl magnetic hydrophilic streptavidin beads (New England Biolabs) were washed three times with 50 μl buffer WB (0.5M NaCl, 20 mM Tris-HCl pH 7.5, 1.0 mM EDTA), resuspended in 5.0 μl TE + 0.3M NaCl and added to each sample.

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    New England Biolabs magnetic hydrophilic streptavidin beads
    Overview of 3-day generation of cDNA libraries. ( A ) On the first day, total RNA is ligated to a 3′ adapter and cDNA is generated by reverse transcription by tandem reactions in a single tube, RNA is degraded and cDNAs are isolated by ethanol precipitation. ( B ) On the second day, cDNAs are circularized, size selected by gel fractionation and eluted overnight in the presence of <t>streptavidin</t> beads. ( C ) PCR is done on bead-bound purified cDNAs to generate templates ready for high-throughput sequencing.
    Magnetic Hydrophilic Streptavidin Beads, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/magnetic hydrophilic streptavidin beads/product/New England Biolabs
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    magnetic hydrophilic streptavidin beads - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

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    Overview of 3-day generation of cDNA libraries. ( A ) On the first day, total RNA is ligated to a 3′ adapter and cDNA is generated by reverse transcription by tandem reactions in a single tube, RNA is degraded and cDNAs are isolated by ethanol precipitation. ( B ) On the second day, cDNAs are circularized, size selected by gel fractionation and eluted overnight in the presence of streptavidin beads. ( C ) PCR is done on bead-bound purified cDNAs to generate templates ready for high-throughput sequencing.

    Journal: Nucleic Acids Research

    Article Title: An efficient and sensitive method for preparing cDNA libraries from scarce biological samples

    doi: 10.1093/nar/gku637

    Figure Lengend Snippet: Overview of 3-day generation of cDNA libraries. ( A ) On the first day, total RNA is ligated to a 3′ adapter and cDNA is generated by reverse transcription by tandem reactions in a single tube, RNA is degraded and cDNAs are isolated by ethanol precipitation. ( B ) On the second day, cDNAs are circularized, size selected by gel fractionation and eluted overnight in the presence of streptavidin beads. ( C ) PCR is done on bead-bound purified cDNAs to generate templates ready for high-throughput sequencing.

    Article Snippet: 5.0 μl magnetic hydrophilic streptavidin beads (New England Biolabs) were washed three times with 50 μl buffer WB (0.5M NaCl, 20 mM Tris-HCl pH 7.5, 1.0 mM EDTA), resuspended in 5.0 μl TE + 0.3M NaCl and added to each sample.

    Techniques: Generated, Isolation, Ethanol Precipitation, Fractionation, Polymerase Chain Reaction, Purification, Next-Generation Sequencing

    Detailed LQ cloning method. ( A ) A pre-adenylated (rApp) 3′-terminal dideoxy-C (ddC) blocked adapter (gray) is annealed to a ssDNA reverse transcription (RT) oligo (black) in a 1:1 molar ratio. The annealed adapter is ligated to 3′-hydroxyl-containing RNA (orange) using T4 RNA Ligase 2 (truncated K227Q) without ATP. Each RT oligo contains a 5′ Guanine (G) followed by a 4 or 6 nucleotide randomer (N X ), a 3–6 nucleotide barcode (BAR) and 3 internal deoxyUridine (dU) nucleotides. The adapter::RT oligo hybrid is in excess over RNA, resulting in free adapter::primer material present in the completed reaction. ( B ) Reverse transcription of ligated RNA is carried out in the same tube as the ligation reaction generating ‘+ insert’ and ‘no insert’ cDNA products (red and black line) using dGTP, dTTP, dATP, dCTP as well as biotinylated dATP and dCTP (yellow ‘B’-containing circles). The RNA template is degraded (dashed orange line) by base hydrolysis and cDNA is ethanol precipitated with ammonium acetate to facilitate maximum removal of free adapter and unincorporated nucleotides ( C ). Ethanol precipitated cDNAs are circularized ( D ) and resolved on a 10% denaturing polyacrylamide gel. ‘+ insert’ circularized cDNAs are isolated by excising and eluting them from the gel overnight in the presence of magnetic streptavidin beads ( E ). Bead-bound ‘+ insert’ cDNAs serve as templates in the first round of PCR. Amplification is done using a mix containing uracil-N-deglycosylase (UNG) to remove dU nucleotides, thereby generating a linear template through strand scission, and with primers complimentary to the 3′ adapter (blue) and 5′ end of the RT oligo (tan) ( F ). First round PCR products are resolved on an 8% native polyacrylamide gel, the 60–70 nucleotide products are excised and a portion is used as the template for second round PCR. Second round PCR products are generated using primers complimentary to the 3′ adapter (dark blue) and 5′ end of the RT oligo (brown) that contain the full Illumina or Ion Torrent adapter sequences (dark blue and brown) ( G ).

    Journal: Nucleic Acids Research

    Article Title: An efficient and sensitive method for preparing cDNA libraries from scarce biological samples

    doi: 10.1093/nar/gku637

    Figure Lengend Snippet: Detailed LQ cloning method. ( A ) A pre-adenylated (rApp) 3′-terminal dideoxy-C (ddC) blocked adapter (gray) is annealed to a ssDNA reverse transcription (RT) oligo (black) in a 1:1 molar ratio. The annealed adapter is ligated to 3′-hydroxyl-containing RNA (orange) using T4 RNA Ligase 2 (truncated K227Q) without ATP. Each RT oligo contains a 5′ Guanine (G) followed by a 4 or 6 nucleotide randomer (N X ), a 3–6 nucleotide barcode (BAR) and 3 internal deoxyUridine (dU) nucleotides. The adapter::RT oligo hybrid is in excess over RNA, resulting in free adapter::primer material present in the completed reaction. ( B ) Reverse transcription of ligated RNA is carried out in the same tube as the ligation reaction generating ‘+ insert’ and ‘no insert’ cDNA products (red and black line) using dGTP, dTTP, dATP, dCTP as well as biotinylated dATP and dCTP (yellow ‘B’-containing circles). The RNA template is degraded (dashed orange line) by base hydrolysis and cDNA is ethanol precipitated with ammonium acetate to facilitate maximum removal of free adapter and unincorporated nucleotides ( C ). Ethanol precipitated cDNAs are circularized ( D ) and resolved on a 10% denaturing polyacrylamide gel. ‘+ insert’ circularized cDNAs are isolated by excising and eluting them from the gel overnight in the presence of magnetic streptavidin beads ( E ). Bead-bound ‘+ insert’ cDNAs serve as templates in the first round of PCR. Amplification is done using a mix containing uracil-N-deglycosylase (UNG) to remove dU nucleotides, thereby generating a linear template through strand scission, and with primers complimentary to the 3′ adapter (blue) and 5′ end of the RT oligo (tan) ( F ). First round PCR products are resolved on an 8% native polyacrylamide gel, the 60–70 nucleotide products are excised and a portion is used as the template for second round PCR. Second round PCR products are generated using primers complimentary to the 3′ adapter (dark blue) and 5′ end of the RT oligo (brown) that contain the full Illumina or Ion Torrent adapter sequences (dark blue and brown) ( G ).

    Article Snippet: 5.0 μl magnetic hydrophilic streptavidin beads (New England Biolabs) were washed three times with 50 μl buffer WB (0.5M NaCl, 20 mM Tris-HCl pH 7.5, 1.0 mM EDTA), resuspended in 5.0 μl TE + 0.3M NaCl and added to each sample.

    Techniques: Clone Assay, Ligation, Isolation, Polymerase Chain Reaction, Amplification, Generated

    SMRT-Cappable-seq identifies full-length transcripts in bacteria. a Schema of the SMRT-Cappable-seq methodology. 5′ triphosphorylated transcripts are capped with a desthio-biotinylated (DTB) cap analog and bound to the streptavidin beads to specifically capture primary transcripts starting at TSS. The polyadenylation step (A-tailing) ensures the priming of the anchored poly dT primer for cDNA synthesis at the most 3′end of the transcript. b Integrative Genomics Viewer (IGV) representation of the mapping of SMRT-Cappable-seq reads (top) compared to Illumina RNA-seq reads (bottom) in the mprA locus. Forward oriented reads are labeled in pink, reverse oriented reads are labeled in blue. c Comparison between gene expression level in Read counts Per Kilobase of transcript, per Million mapped reads (RPKM) for Illumina RNA-seq and SMRT-Cappable-seq. The Spearman’s rank correlation is 0.798 ( p value

    Journal: Nature Communications

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria

    doi: 10.1038/s41467-018-05997-6

    Figure Lengend Snippet: SMRT-Cappable-seq identifies full-length transcripts in bacteria. a Schema of the SMRT-Cappable-seq methodology. 5′ triphosphorylated transcripts are capped with a desthio-biotinylated (DTB) cap analog and bound to the streptavidin beads to specifically capture primary transcripts starting at TSS. The polyadenylation step (A-tailing) ensures the priming of the anchored poly dT primer for cDNA synthesis at the most 3′end of the transcript. b Integrative Genomics Viewer (IGV) representation of the mapping of SMRT-Cappable-seq reads (top) compared to Illumina RNA-seq reads (bottom) in the mprA locus. Forward oriented reads are labeled in pink, reverse oriented reads are labeled in blue. c Comparison between gene expression level in Read counts Per Kilobase of transcript, per Million mapped reads (RPKM) for Illumina RNA-seq and SMRT-Cappable-seq. The Spearman’s rank correlation is 0.798 ( p value

    Article Snippet: The capped RNA was enriched using hydrophilic streptavidin magnetic beads (New England Biolabs).

    Techniques: RNA Sequencing Assay, Labeling, Expressing

    Overview of the Capp-Switch sequencing approach. Capp-Switch includes ( a – c ) capture of 5′ mRNA fragments and ( d – f ) cDNA synthesis and sequencing. ( a ) The mRNA 5′ triphosphate is capped with biotin-GTP by VCE. ( b ) RNA is fragmented and ( c ) the capped 5′ mRNA fragments are captured on streptavidin magnetic beads and separated from other RNA. ( d ) The 5′ mRNA fragments are reverse transcribed to single-stranded cDNA using MMLV reverse transcriptase. An oligonucleotide hybridizes to the 3′ overhang and the complementary sequence is synthesized by the MMLV template-switching activity. ( e ) Double-stranded cDNA is synthesized using primers that hybridize to the single-stranded cDNA termini. ( f ) The cDNA is sequenced on a high-throughput platform.

    Journal: Nature Communications

    Article Title: Global repositioning of transcription start sites in a plant-fermenting bacterium

    doi: 10.1038/ncomms13783

    Figure Lengend Snippet: Overview of the Capp-Switch sequencing approach. Capp-Switch includes ( a – c ) capture of 5′ mRNA fragments and ( d – f ) cDNA synthesis and sequencing. ( a ) The mRNA 5′ triphosphate is capped with biotin-GTP by VCE. ( b ) RNA is fragmented and ( c ) the capped 5′ mRNA fragments are captured on streptavidin magnetic beads and separated from other RNA. ( d ) The 5′ mRNA fragments are reverse transcribed to single-stranded cDNA using MMLV reverse transcriptase. An oligonucleotide hybridizes to the 3′ overhang and the complementary sequence is synthesized by the MMLV template-switching activity. ( e ) Double-stranded cDNA is synthesized using primers that hybridize to the single-stranded cDNA termini. ( f ) The cDNA is sequenced on a high-throughput platform.

    Article Snippet: Streptavidin magnetic beads (NEB S1421S) were pre-washed twice with low-salt buffer (10 mM Tris, 50 mM NaCl, 1 mM EDTA), twice with binding buffer (10 mM Tris, 500 mM NaCl, 1 mM EDTA) and resuspended at 4 mg ml−1 beads in binding buffer.

    Techniques: Sequencing, Magnetic Beads, Synthesized, Activity Assay, High Throughput Screening Assay

    Cleavage of the membrane-adjacent extracellular fragment of NCAM2 is increased in AD brains. ( a ) Diagram showing the structure of NCAM2. Ig, immunoglobulin-like domain; Fn, fibronectin type III domain. Peptides used in the cleavage assay and corresponding to aa682-701 (NCAM2aa682-701) and aa666-685 (NCAM2aa666-685) of human NCAM2 are shown below. Asparagine 689 and aspartic acid 693 exchanged to alanine in the cleavage assay shown in c are highlighted in bold. ( b ) Scheme of the peptide cleavage assay. Peptides labelled with FITC and biotin at the N- and C-terminus, respectively, were incubated either with the total brain lysate or lysate of synaptosomes. Non-cleaved peptides and biotin-containing fragments of the cleaved peptides were removed using streptavidin-coated beads. The remaining FITC fluorescence was used as an estimate of peptide cleavage. ( c ) Graphs show the efficiency of the peptide cleavage (mean+s.e.m.) with the fluorescence signals for NCAM2aa682-701 in controls set to 100%. Lysates from eight controls and eight AD patients were analysed. Note that the efficiency of NCAM2aa682-701 cleavage is higher in AD cases and particularly in synaptosomes. NCAM2aa682-701 cleavage is reduced by mutating aspartic acid 693. * P

    Journal: Nature Communications

    Article Title: Aβ-dependent reduction of NCAM2-mediated synaptic adhesion contributes to synapse loss in Alzheimer's disease

    doi: 10.1038/ncomms9836

    Figure Lengend Snippet: Cleavage of the membrane-adjacent extracellular fragment of NCAM2 is increased in AD brains. ( a ) Diagram showing the structure of NCAM2. Ig, immunoglobulin-like domain; Fn, fibronectin type III domain. Peptides used in the cleavage assay and corresponding to aa682-701 (NCAM2aa682-701) and aa666-685 (NCAM2aa666-685) of human NCAM2 are shown below. Asparagine 689 and aspartic acid 693 exchanged to alanine in the cleavage assay shown in c are highlighted in bold. ( b ) Scheme of the peptide cleavage assay. Peptides labelled with FITC and biotin at the N- and C-terminus, respectively, were incubated either with the total brain lysate or lysate of synaptosomes. Non-cleaved peptides and biotin-containing fragments of the cleaved peptides were removed using streptavidin-coated beads. The remaining FITC fluorescence was used as an estimate of peptide cleavage. ( c ) Graphs show the efficiency of the peptide cleavage (mean+s.e.m.) with the fluorescence signals for NCAM2aa682-701 in controls set to 100%. Lysates from eight controls and eight AD patients were analysed. Note that the efficiency of NCAM2aa682-701 cleavage is higher in AD cases and particularly in synaptosomes. NCAM2aa682-701 cleavage is reduced by mutating aspartic acid 693. * P

    Article Snippet: Non-cleaved peptides and biotin-containing fragments of cleaved peptide were removed by incubating lysates with hydrophilic streptavidin magnetic beads (New England BioLabs) for 1 h at room temperature.

    Techniques: Cleavage Assay, Incubation, Fluorescence