tris hcl  (New England Biolabs)


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    Structured Review

    New England Biolabs tris hcl
    Template switching of LtrA from the 5′ end of the Ll.LtrB intron RNA to exon 1 DNA or RNA. The Ll.LtrB intron RT (LtrA protein; 40 nM) was incubated with artificial substrates corresponding to the 5′ end of Ll.LtrB intron (Ll.LtrB RNA; 40 nM) with an annealed 5′- 32 P-labeled DNA primer c (Pri c; 44 nM) in presence of exon 1 (E1) DNA or RNA (40 nM; black and red, respectively), as diagrammed in schematics to the left of the gel. The substrates were incubated with dNTPs (200 µM) in reaction medium containing 450 mM <t>NaCl,</t> 5 mM MgCl 2 , 20 mM <t>Tris-HCl,</t> pH 7.5, and 1 mM DTT for 30 min at 30°C. After terminating the reaction by extraction with phenol-CIA, the products were analyzed in a denaturing 15% polyacrylamide gel. Lanes (1) and (2) 32 P-labeled Pri c incubated without and with LtrA, respectively; (3) and (4) LtrA incubated with 32 P-labeled Pri c and E1 DNA or RNA, respectively; (5) and (6) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA, respectively; (7–9) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA with annealed complementary DNA oligonucleotides to leave a blunt end (exon 1 AS) or a 5′-bottom-strand overhang (exon 1 AS+9). Bands excised for sequencing ( Figure 7 ) are indicated in the gel. In the schematics, DNA and RNA oligonucleotides are shown in black and red, respectively; LtrA is shown as a gray oval; and the direction of cDNA synthesis is indicated by a green arrow. The numbers to the right of the gel indicate the positions of 5′-end labeled size markers (10-bp DNA ladder, Invitrogen).
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    Images

    1) Product Images from "The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4-Dependent and -Independent Mechanisms"

    Article Title: The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4-Dependent and -Independent Mechanisms

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002534

    Template switching of LtrA from the 5′ end of the Ll.LtrB intron RNA to exon 1 DNA or RNA. The Ll.LtrB intron RT (LtrA protein; 40 nM) was incubated with artificial substrates corresponding to the 5′ end of Ll.LtrB intron (Ll.LtrB RNA; 40 nM) with an annealed 5′- 32 P-labeled DNA primer c (Pri c; 44 nM) in presence of exon 1 (E1) DNA or RNA (40 nM; black and red, respectively), as diagrammed in schematics to the left of the gel. The substrates were incubated with dNTPs (200 µM) in reaction medium containing 450 mM NaCl, 5 mM MgCl 2 , 20 mM Tris-HCl, pH 7.5, and 1 mM DTT for 30 min at 30°C. After terminating the reaction by extraction with phenol-CIA, the products were analyzed in a denaturing 15% polyacrylamide gel. Lanes (1) and (2) 32 P-labeled Pri c incubated without and with LtrA, respectively; (3) and (4) LtrA incubated with 32 P-labeled Pri c and E1 DNA or RNA, respectively; (5) and (6) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA, respectively; (7–9) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA with annealed complementary DNA oligonucleotides to leave a blunt end (exon 1 AS) or a 5′-bottom-strand overhang (exon 1 AS+9). Bands excised for sequencing ( Figure 7 ) are indicated in the gel. In the schematics, DNA and RNA oligonucleotides are shown in black and red, respectively; LtrA is shown as a gray oval; and the direction of cDNA synthesis is indicated by a green arrow. The numbers to the right of the gel indicate the positions of 5′-end labeled size markers (10-bp DNA ladder, Invitrogen).
    Figure Legend Snippet: Template switching of LtrA from the 5′ end of the Ll.LtrB intron RNA to exon 1 DNA or RNA. The Ll.LtrB intron RT (LtrA protein; 40 nM) was incubated with artificial substrates corresponding to the 5′ end of Ll.LtrB intron (Ll.LtrB RNA; 40 nM) with an annealed 5′- 32 P-labeled DNA primer c (Pri c; 44 nM) in presence of exon 1 (E1) DNA or RNA (40 nM; black and red, respectively), as diagrammed in schematics to the left of the gel. The substrates were incubated with dNTPs (200 µM) in reaction medium containing 450 mM NaCl, 5 mM MgCl 2 , 20 mM Tris-HCl, pH 7.5, and 1 mM DTT for 30 min at 30°C. After terminating the reaction by extraction with phenol-CIA, the products were analyzed in a denaturing 15% polyacrylamide gel. Lanes (1) and (2) 32 P-labeled Pri c incubated without and with LtrA, respectively; (3) and (4) LtrA incubated with 32 P-labeled Pri c and E1 DNA or RNA, respectively; (5) and (6) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA, respectively; (7–9) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA with annealed complementary DNA oligonucleotides to leave a blunt end (exon 1 AS) or a 5′-bottom-strand overhang (exon 1 AS+9). Bands excised for sequencing ( Figure 7 ) are indicated in the gel. In the schematics, DNA and RNA oligonucleotides are shown in black and red, respectively; LtrA is shown as a gray oval; and the direction of cDNA synthesis is indicated by a green arrow. The numbers to the right of the gel indicate the positions of 5′-end labeled size markers (10-bp DNA ladder, Invitrogen).

    Techniques Used: Incubation, Labeling, Sequencing

    2) Product Images from "Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase"

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl060

    Size-exclusion chromatographic analysis of wild-type and S326C OGG1. ( A ) Non-denatured protein size markers (Sigma). Peak 1, BSA dimer (132 kDa); peak 2, BSA monomer (66 kDa) and peak 3, carbonic anydrase (29 kDa). Purified wild-type OGG1 (100 µg) was analyzed on a Superdex 200 HR column equilibrated with 20 mM Tris–HCl (pH 7.4), 300 mM NaCl at a flow rate of 0.25 ml/min ( B ). Identical runs were performed with 100 µg polymorphic S326C OGG1 ( C ) or 100 µg of both wild-type and S326C OGG1 together ( D ).
    Figure Legend Snippet: Size-exclusion chromatographic analysis of wild-type and S326C OGG1. ( A ) Non-denatured protein size markers (Sigma). Peak 1, BSA dimer (132 kDa); peak 2, BSA monomer (66 kDa) and peak 3, carbonic anydrase (29 kDa). Purified wild-type OGG1 (100 µg) was analyzed on a Superdex 200 HR column equilibrated with 20 mM Tris–HCl (pH 7.4), 300 mM NaCl at a flow rate of 0.25 ml/min ( B ). Identical runs were performed with 100 µg polymorphic S326C OGG1 ( C ) or 100 µg of both wild-type and S326C OGG1 together ( D ).

    Techniques Used: Purification, Flow Cytometry

    Related Articles

    DNA Extraction:

    Article Title: MiRAR—miRNA Activity Reporter for Living Cells
    Article Snippet: Paragraph title: 2.1. Genomic DNA Extraction ... Cells from half of a 150 mm plate (~107 cells) were resuspended in 2.4 mL lysis buffer (0.6% sodium dodecyl sulfate (SDS), 10 mM ethylenediaminetetraacetic acid (EDTA), 10 mM Tris-HCl pH 8.0) and 25 units RNase I (NEB M0243S) and incubated at 37 °C for 1 h. This was followed by the addition of 240 µL 5 M NaCl (6 mmol) and 1 h of incubation on ice.

    Centrifugation:

    Article Title: The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4-Dependent and -Independent Mechanisms
    Article Snippet: After pelleting cell debris (Beckman JA-14 rotor, 10,000 rpm, 30 min, 4°C), nucleic acids were precipitated from the supernatant with 0.4% polyethylenimine (PEI) and constant stirring for 20 min at 4°C, followed by centrifugation (Beckman JA-14 rotor, 14,000 rpm, 30 min, 4°C). .. The precipitated proteins were pelleted (Beckman JA-14 rotor, 14,000 rpm, 30 min, 4°C), dissolved in 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol, and run through a 10-ml amylose column (FPLC; Amylose High-Flow resin; New England BioLabs, Ipswich, MA), which was washed with 3 column volumes of 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol and eluted with 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol containing 10 mM maltose.

    Amplification:

    Article Title: MiRAR—miRNA Activity Reporter for Living Cells
    Article Snippet: Genomic DNA was prepared as a template for the amplification of 3′-UTRs as described previously [ ]. .. Cells from half of a 150 mm plate (~107 cells) were resuspended in 2.4 mL lysis buffer (0.6% sodium dodecyl sulfate (SDS), 10 mM ethylenediaminetetraacetic acid (EDTA), 10 mM Tris-HCl pH 8.0) and 25 units RNase I (NEB M0243S) and incubated at 37 °C for 1 h. This was followed by the addition of 240 µL 5 M NaCl (6 mmol) and 1 h of incubation on ice.

    Synthesized:

    Article Title: A Phenolic Fraction from Catharanthus roseus L. Stems Decreases Glycemia and Stimulates Insulin Secretion
    Article Snippet: The cDNA was synthesized by retrotranscription using the enzyme M-MuLV-RT (New-England Biolabs™, USA), following the manufacturer's instructions. .. The reaction was carried out with 1 μ g of RNA in a final volume of 20 μ L. The reaction mixture contained 4 μ M hexamers (Fermentas™, UK), 1X Reverse Transcriptase Buffer 75 mM KCl, 50 mM Tris-HCl, 3 mM MgCl2, 10 mM dithiothreitol (New-England Biolabs™, USA), 10 μ M dNTPs (Promega®, USA), 20 U RNase Inhibitor (Ambion™, USA), and 100 U MuLV Reverse Transcriptase (New-England Biolabs™, USA).

    Quantitative RT-PCR:

    Article Title: A Phenolic Fraction from Catharanthus roseus L. Stems Decreases Glycemia and Stimulates Insulin Secretion
    Article Snippet: The reaction was carried out with 1 μ g of RNA in a final volume of 20 μ L. The reaction mixture contained 4 μ M hexamers (Fermentas™, UK), 1X Reverse Transcriptase Buffer 75 mM KCl, 50 mM Tris-HCl, 3 mM MgCl2, 10 mM dithiothreitol (New-England Biolabs™, USA), 10 μ M dNTPs (Promega®, USA), 20 U RNase Inhibitor (Ambion™, USA), and 100 U MuLV Reverse Transcriptase (New-England Biolabs™, USA). .. The reaction was carried out with 1 μ g of RNA in a final volume of 20 μ L. The reaction mixture contained 4 μ M hexamers (Fermentas™, UK), 1X Reverse Transcriptase Buffer 75 mM KCl, 50 mM Tris-HCl, 3 mM MgCl2, 10 mM dithiothreitol (New-England Biolabs™, USA), 10 μ M dNTPs (Promega®, USA), 20 U RNase Inhibitor (Ambion™, USA), and 100 U MuLV Reverse Transcriptase (New-England Biolabs™, USA).

    Real-time Polymerase Chain Reaction:

    Article Title: A Phenolic Fraction from Catharanthus roseus L. Stems Decreases Glycemia and Stimulates Insulin Secretion
    Article Snippet: The reaction was carried out with 1 μ g of RNA in a final volume of 20 μ L. The reaction mixture contained 4 μ M hexamers (Fermentas™, UK), 1X Reverse Transcriptase Buffer 75 mM KCl, 50 mM Tris-HCl, 3 mM MgCl2, 10 mM dithiothreitol (New-England Biolabs™, USA), 10 μ M dNTPs (Promega®, USA), 20 U RNase Inhibitor (Ambion™, USA), and 100 U MuLV Reverse Transcriptase (New-England Biolabs™, USA). .. The reaction was carried out with 1 μ g of RNA in a final volume of 20 μ L. The reaction mixture contained 4 μ M hexamers (Fermentas™, UK), 1X Reverse Transcriptase Buffer 75 mM KCl, 50 mM Tris-HCl, 3 mM MgCl2, 10 mM dithiothreitol (New-England Biolabs™, USA), 10 μ M dNTPs (Promega®, USA), 20 U RNase Inhibitor (Ambion™, USA), and 100 U MuLV Reverse Transcriptase (New-England Biolabs™, USA).

    Incubation:

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: Unincorporated radioactivity was removed by using MicroSpin G-25 columns (Amersham). .. OGG1 proteins or nuclear extracts were incubated with DNA substrates at varying concentrations in 20 µl reactions containing 20 mM Tris–HCl (pH 7.4), 100 mM NaCl and 0.15 µg/µl BSA (New England Biolabs). .. To measure glycosylase activity, reactions were terminated by adding SDS and piperidine to 5% and 200 mM, respectively.

    Article Title: Cotranslational assembly of protein complexes in eukaryotes revealed by ribosome profiling
    Article Snippet: For linker ligation, a maximum of 5 pmol RNA in 5 µl were denatured for 2 min at 80°C before 8 µl 50% sterile filtered PEG MW 8000, 2 µl DMSO, 2 µl 10x T4 RNA Ligase 2 buffer (NEB), 1 µl murine RNase inhibitor, 1 µl 1 mg/µl linker L1 and 1 µl truncated T4 RNA Ligase 2 (NEB) were added and incubated for 2.5 hr at 37°C or 23°C. .. For reverse transcription, RNA was resuspended in 10 µl 10 mM Tris-HCl pH 7.0 and 1 µl 10 mM dNTP (NEB), 1 µl 25 linker L1’L20 and 1.5µl DEPC H20 were added to each sample.

    Article Title: DISC1-dependent switch from progenitor proliferation to migration in the developing cortex
    Article Snippet: Soluble proteins obtained from the cells overexpressing myc-tagged wild-type human DISC1 (hDISC1) were incubated with an antibody against myc-tag (Santa Cruz) and Protein G Plus/Protein A agarose beads (Calbiochem) at 4 °C overnight. .. Beads were washed in 20 mM Tris-HCl, pH 7.6, three times and in lambda phosphatase buffer (New England Biolabs) once, and phosphatase reactions were performed directly on the beads at 30°C for 2 h with lambda phosphatase (New England Biolabs) as per manufacturer's protocol.

    Article Title: Stress-Induced Low Complexity RNA Activates Physiological Amyloidogenesis
    Article Snippet: Post-fixation for 30 min, cells were quenched with 0.1M Tris-HCl, pH 7.0 for 10 min before Proteinase K (PK) treatment (NEB, 800U/ml stock, 100,000× dilution) at 37°C for 30 min. PK treatment was followed by a 5-min 1% formaldehyde post-fix. .. Cells were blocked for 30 min with 5% horse serum in PBS followed by incubation with primary antibody rabbit anti-A11 (1:100) for 1 h at 37°C.

    Article Title: MiRAR—miRNA Activity Reporter for Living Cells
    Article Snippet: Briefly, human embryonic kidney 293 (HEK 293) cells were grown to confluency in Dulbecco’s modified eagle medium (DMEM) from Gibco (Thermo Fisher Scientific, Waltham, MA, USA) and 10% fetal bovine serum (FBS). .. Cells from half of a 150 mm plate (~107 cells) were resuspended in 2.4 mL lysis buffer (0.6% sodium dodecyl sulfate (SDS), 10 mM ethylenediaminetetraacetic acid (EDTA), 10 mM Tris-HCl pH 8.0) and 25 units RNase I (NEB M0243S) and incubated at 37 °C for 1 h. This was followed by the addition of 240 µL 5 M NaCl (6 mmol) and 1 h of incubation on ice. .. The solution was centrifuged at 10,000× g for 30 min at 4 °C and the DNA was extracted from the supernatant via phenol chloroform extraction.

    Article Title: Lysosomal membrane protein SIDT2 mediates the direct uptake of DNA by lysosomes
    Article Snippet: Intact isolated lysosomes were incubated with plasmid DNA in the presence of an ATP regeneration system for 5 min at 37°C. .. Following incubation, DNA was extracted as follows: A proteinase K solution containing 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% SDS, and 0.1 mg/ml proteinase K (New England Biolabs Inc., P8102S) in saline sodium citrate buffer was added and incubated for 2 h at 37°C, followed by phenol-chloroform extraction and ethanol precipitation. .. Total levels of DNA were analyzed by agarose gel electrophoresis with ethidium bromide staining and UV illumination.

    Article Title: Restoration of Compact Golgi Morphology in Advanced Prostate Cancer Enhances Susceptibility to Galectin-1-induced Apoptosis by Modifying Mucin O-glycan Synthesis
    Article Snippet: Cell samples were resuspended in EDTA-free CIP buffer, containing 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , and 1 mM DTT (New England Biolabs). .. Cell samples were resuspended in EDTA-free CIP buffer, containing 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , and 1 mM DTT (New England Biolabs).

    Article Title: Selection of Single-Stranded DNA Molecular Recognition Elements against Exotoxin A Using a Novel Decoy-SELEX Method and Sensitive Detection of Exotoxin A in Human Serum
    Article Snippet: Immobilization substrate was prepared by incubating Sulfo-NHS-LC-Biotin (Pierce; Rockford, IL, USA) with Tris-HCl and streptavidin-coated magnetic beads (New England Biolabs, Ipswich, MA, USA). .. After magnetic separation, unbound ssDNA served as the template for PCR amplification as illustrated above.

    Article Title: Adaptation to DNA damage checkpoint in senescent telomerase-negative cells promotes genome instability
    Article Snippet: Plugs were incubated in the same buffer containing 10 mM dithiothreitol and 0.4 mg/mL Zymolyase 20T (Seikagaku) for 24 h at 37°C. .. The solution was then replaced with 10 mM Tris-HCl (pH 8.0) containing 50 mM EDTA, 1% sarkosyl, and 2 mg/mL proteinase K (New England Biolabs), and the plugs were incubated for 24 h at 50°C. .. After extensive washing with 10 mM Tris-HCl and 50 mM EDTA (pH 8.0), the plugs were loaded into the wells of a 0.9% agarose gel (Seakem Gold, Ozyme) and electrophoresed at 13°C in a rotating PFGE apparatus (Rotaphor 6.0, Biometra) according to the manufacturer's instructions for separation of S. cerevisiae chromosomes.

    Article Title: A Phenolic Fraction from Catharanthus roseus L. Stems Decreases Glycemia and Stimulates Insulin Secretion
    Article Snippet: After 24 h, the medium was replaced with a medium containing the C. roseus precipitate (10 and 100 μ g/mL) and incubated for 1, 2, 4, 8, 16, and 24 h. Cells were then washed with PBS. .. The reaction was carried out with 1 μ g of RNA in a final volume of 20 μ L. The reaction mixture contained 4 μ M hexamers (Fermentas™, UK), 1X Reverse Transcriptase Buffer 75 mM KCl, 50 mM Tris-HCl, 3 mM MgCl2, 10 mM dithiothreitol (New-England Biolabs™, USA), 10 μ M dNTPs (Promega®, USA), 20 U RNase Inhibitor (Ambion™, USA), and 100 U MuLV Reverse Transcriptase (New-England Biolabs™, USA).

    Activity Assay:

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: Paragraph title: 8-oxoguanine glycosylase and AP-lyase activity assays ... OGG1 proteins or nuclear extracts were incubated with DNA substrates at varying concentrations in 20 µl reactions containing 20 mM Tris–HCl (pH 7.4), 100 mM NaCl and 0.15 µg/µl BSA (New England Biolabs).

    Article Title: MDP: A Deinococcus Mn2+-Decapeptide Complex Protects Mice from Ionizing Radiation
    Article Snippet: Typically, 50 μl of the T4 DNA ligase mixtures were irradiated with 60 Co at 12 kGy/h (Shepherd and Associates model 109–68 irradiator) aerobically on ice. .. Following irradiation, 5 μl of each IR-treated T4 DNA ligase sample were assayed for residual ligase activity in separate reaction mixtures (final volume, 50 μl) containing 300 ng of Xba I-linearized pUC19 DNA, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 10 mM dithiothreitol and 1 mM ATP (New England Biolabs). .. T4 DNA ligase/pUC19 DNA mixtures were incubated for 16 h at 16°C, followed by agarose (1%) gel electrophoresis.

    Expressing:

    Article Title: Stress-Induced Low Complexity RNA Activates Physiological Amyloidogenesis
    Article Snippet: Expression levels were normalized to account for fewer input reads in the 30 min heat shock sample (after rRNA sequences were discarded). .. Post-fixation of 30’, cells were quenched with 0.1M Tris-HCl, pH 7.0 for 10’ before ± Proteinase K (PK) treatment (NEB, 800U/ml stock, 100,000× dilution) at 37°C for 30’.

    Article Title: A Phenolic Fraction from Catharanthus roseus L. Stems Decreases Glycemia and Stimulates Insulin Secretion
    Article Snippet: Paragraph title: 2.3.2. Expression of Insulin mRNA in RINm5F Cells Treated with the Precipitate from C. roseus ... The reaction was carried out with 1 μ g of RNA in a final volume of 20 μ L. The reaction mixture contained 4 μ M hexamers (Fermentas™, UK), 1X Reverse Transcriptase Buffer 75 mM KCl, 50 mM Tris-HCl, 3 mM MgCl2, 10 mM dithiothreitol (New-England Biolabs™, USA), 10 μ M dNTPs (Promega®, USA), 20 U RNase Inhibitor (Ambion™, USA), and 100 U MuLV Reverse Transcriptase (New-England Biolabs™, USA).

    Modification:

    Article Title: MiRAR—miRNA Activity Reporter for Living Cells
    Article Snippet: Briefly, human embryonic kidney 293 (HEK 293) cells were grown to confluency in Dulbecco’s modified eagle medium (DMEM) from Gibco (Thermo Fisher Scientific, Waltham, MA, USA) and 10% fetal bovine serum (FBS). .. Cells from half of a 150 mm plate (~107 cells) were resuspended in 2.4 mL lysis buffer (0.6% sodium dodecyl sulfate (SDS), 10 mM ethylenediaminetetraacetic acid (EDTA), 10 mM Tris-HCl pH 8.0) and 25 units RNase I (NEB M0243S) and incubated at 37 °C for 1 h. This was followed by the addition of 240 µL 5 M NaCl (6 mmol) and 1 h of incubation on ice.

    Western Blot:

    Article Title: DISC1-dependent switch from progenitor proliferation to migration in the developing cortex
    Article Snippet: Beads were washed in 20 mM Tris-HCl, pH 7.6, three times and in lambda phosphatase buffer (New England Biolabs) once, and phosphatase reactions were performed directly on the beads at 30°C for 2 h with lambda phosphatase (New England Biolabs) as per manufacturer's protocol. .. Mouse brains were homogenized in lysis buffer.

    Hybridization:

    Article Title: Stress-Induced Low Complexity RNA Activates Physiological Amyloidogenesis
    Article Snippet: Post-fixation of 30’, cells were quenched with 0.1M Tris-HCl, pH 7.0 for 10’ before ± Proteinase K (PK) treatment (NEB, 800U/ml stock, 100,000× dilution) at 37°C for 30’. .. Post-fixation of 30’, cells were quenched with 0.1M Tris-HCl, pH 7.0 for 10’ before ± Proteinase K (PK) treatment (NEB, 800U/ml stock, 100,000× dilution) at 37°C for 30’.

    Ligation:

    Article Title: Cotranslational assembly of protein complexes in eukaryotes revealed by ribosome profiling
    Article Snippet: For linker ligation, a maximum of 5 pmol RNA in 5 µl were denatured for 2 min at 80°C before 8 µl 50% sterile filtered PEG MW 8000, 2 µl DMSO, 2 µl 10x T4 RNA Ligase 2 buffer (NEB), 1 µl murine RNase inhibitor, 1 µl 1 mg/µl linker L1 and 1 µl truncated T4 RNA Ligase 2 (NEB) were added and incubated for 2.5 hr at 37°C or 23°C. .. For reverse transcription, RNA was resuspended in 10 µl 10 mM Tris-HCl pH 7.0 and 1 µl 10 mM dNTP (NEB), 1 µl 25 linker L1’L20 and 1.5µl DEPC H20 were added to each sample.

    In Situ Hybridization:

    Article Title: Stress-Induced Low Complexity RNA Activates Physiological Amyloidogenesis
    Article Snippet: In situ hybridization was carried out with 5’ and 3’ digoxigenin (DIG)-labeled oligonucleotides. .. Post-fixation of 30’, cells were quenched with 0.1M Tris-HCl, pH 7.0 for 10’ before ± Proteinase K (PK) treatment (NEB, 800U/ml stock, 100,000× dilution) at 37°C for 30’.

    other:

    Article Title: Altered DNA Binding and Amplification of Human Breast Cancer Suppressor Gene BRCA1 Induced by a Novel Antitumor Compound, [Ru(?6-p-phenylethacrynate)Cl2(pta)]
    Article Snippet: Taq DNA polymerase, Pvu II, Eco O190I, dNTPs and Tris-HCl were from New England Biolabs.

    Sequencing:

    Article Title: Cotranslational assembly of protein complexes in eukaryotes revealed by ribosome profiling
    Article Snippet: Paragraph title: cDNA library preparation for deep sequencing ... For reverse transcription, RNA was resuspended in 10 µl 10 mM Tris-HCl pH 7.0 and 1 µl 10 mM dNTP (NEB), 1 µl 25 linker L1’L20 and 1.5µl DEPC H20 were added to each sample.

    Article Title: Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers
    Article Snippet: Small RNA was first ligated to 3′ DNA adapters with adenylated 5′ and dideoxycytosine-blocked 3′ ends in 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 10 mM DTT, and 50% PEG 8000 (New England Biolabs, Ipswich, MA, USA) with T4 Rnl2tr K227Q (homemade) at 25 °C for 16 h. The 3′ adapter contained UMIs in 3 nt-blocks of random nucleotides separated by pre-defined 3 nt consensus sequences (NNN-GTC-NNN-TAG-NNN, Fig. ). .. The 54–71 nt (18–35 nt small RNA + 36 nt 3′ UMI adapter) 3′ ligated product was purified from a 10% denaturing urea-polyacrylamide gel.

    Sonication:

    Article Title: The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4-Dependent and -Independent Mechanisms
    Article Snippet: Cells were harvested by centrifugation (Beckman JLA-8.1000; 4,000× g, 15 min, 4°C), resuspended in 1 M NaCl, 20 mM Tris-HCl, pH 7.5, 20% glycerol, and 0.1 mg/ml lysozyme (Sigma-Aldrich, St. Louis, MO), kept on ice for 15 min, and lysed by 3 freeze-thaw cycles on dry ice followed by sonication (Branson 450 Sonifier, Branson Ultrasonics, Danbury, CT; three or four 10 sec bursts on ice at an amplitude of 60%, with 10 sec between bursts). .. The precipitated proteins were pelleted (Beckman JA-14 rotor, 14,000 rpm, 30 min, 4°C), dissolved in 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol, and run through a 10-ml amylose column (FPLC; Amylose High-Flow resin; New England BioLabs, Ipswich, MA), which was washed with 3 column volumes of 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol and eluted with 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol containing 10 mM maltose.

    Multiplexing:

    Article Title: Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers
    Article Snippet: Small RNA was first ligated to 3′ DNA adapters with adenylated 5′ and dideoxycytosine-blocked 3′ ends in 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 10 mM DTT, and 50% PEG 8000 (New England Biolabs, Ipswich, MA, USA) with T4 Rnl2tr K227Q (homemade) at 25 °C for 16 h. The 3′ adapter contained UMIs in 3 nt-blocks of random nucleotides separated by pre-defined 3 nt consensus sequences (NNN-GTC-NNN-TAG-NNN, Fig. ). .. The 54–71 nt (18–35 nt small RNA + 36 nt 3′ UMI adapter) 3′ ligated product was purified from a 10% denaturing urea-polyacrylamide gel.

    Radioactivity:

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: OGG1 proteins or nuclear extracts were incubated with DNA substrates at varying concentrations in 20 µl reactions containing 20 mM Tris–HCl (pH 7.4), 100 mM NaCl and 0.15 µg/µl BSA (New England Biolabs). .. When measuring AP-lyase activity with an AP site substrate, reactions were terminated by adding SDS and glycerol to 5 and 10%, respectively, without heating.

    RNA Sequencing Assay:

    Article Title: Stress-Induced Low Complexity RNA Activates Physiological Amyloidogenesis
    Article Snippet: For the graphical representation of the RNA-seq data, base coverage profiles of GL000220.1, an unplaced contig containing an entire ribosomal cassette, were produced in Integrative Genomics Viewer. .. Post-fixation of 30’, cells were quenched with 0.1M Tris-HCl, pH 7.0 for 10’ before ± Proteinase K (PK) treatment (NEB, 800U/ml stock, 100,000× dilution) at 37°C for 30’.

    Article Title: Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers
    Article Snippet: Paragraph title: Small RNA-seq library construction ... Small RNA was first ligated to 3′ DNA adapters with adenylated 5′ and dideoxycytosine-blocked 3′ ends in 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 10 mM DTT, and 50% PEG 8000 (New England Biolabs, Ipswich, MA, USA) with T4 Rnl2tr K227Q (homemade) at 25 °C for 16 h. The 3′ adapter contained UMIs in 3 nt-blocks of random nucleotides separated by pre-defined 3 nt consensus sequences (NNN-GTC-NNN-TAG-NNN, Fig. ).

    Magnetic Beads:

    Article Title: Selection of Single-Stranded DNA Molecular Recognition Elements against Exotoxin A Using a Novel Decoy-SELEX Method and Sensitive Detection of Exotoxin A in Human Serum
    Article Snippet: Round 1(−) was performed by incubating enriched ssDNA from the preceding positive round with immobilization substrate in a total volume of 100 μ L selection buffer at room temperature for 18 hours on rotisserie. .. Immobilization substrate was prepared by incubating Sulfo-NHS-LC-Biotin (Pierce; Rockford, IL, USA) with Tris-HCl and streptavidin-coated magnetic beads (New England Biolabs, Ipswich, MA, USA). .. After magnetic separation, unbound ssDNA served as the template for PCR amplification as illustrated above.

    Isolation:

    Article Title: Lysosomal membrane protein SIDT2 mediates the direct uptake of DNA by lysosomes
    Article Snippet: Intact isolated lysosomes were incubated with plasmid DNA in the presence of an ATP regeneration system for 5 min at 37°C. .. Following incubation, DNA was extracted as follows: A proteinase K solution containing 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% SDS, and 0.1 mg/ml proteinase K (New England Biolabs Inc., P8102S) in saline sodium citrate buffer was added and incubated for 2 h at 37°C, followed by phenol-chloroform extraction and ethanol precipitation.

    Article Title: Restoration of Compact Golgi Morphology in Advanced Prostate Cancer Enhances Susceptibility to Galectin-1-induced Apoptosis by Modifying Mucin O-glycan Synthesis
    Article Snippet: Paragraph title: Phosphorylation and the plasma membrane protein isolation assay ... Cell samples were resuspended in EDTA-free CIP buffer, containing 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , and 1 mM DTT (New England Biolabs).

    Size-exclusion Chromatography:

    Article Title: The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4-Dependent and -Independent Mechanisms
    Article Snippet: Cells were harvested by centrifugation (Beckman JLA-8.1000; 4,000× g, 15 min, 4°C), resuspended in 1 M NaCl, 20 mM Tris-HCl, pH 7.5, 20% glycerol, and 0.1 mg/ml lysozyme (Sigma-Aldrich, St. Louis, MO), kept on ice for 15 min, and lysed by 3 freeze-thaw cycles on dry ice followed by sonication (Branson 450 Sonifier, Branson Ultrasonics, Danbury, CT; three or four 10 sec bursts on ice at an amplitude of 60%, with 10 sec between bursts). .. The precipitated proteins were pelleted (Beckman JA-14 rotor, 14,000 rpm, 30 min, 4°C), dissolved in 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol, and run through a 10-ml amylose column (FPLC; Amylose High-Flow resin; New England BioLabs, Ipswich, MA), which was washed with 3 column volumes of 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol and eluted with 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol containing 10 mM maltose.

    Purification:

    Article Title: The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4-Dependent and -Independent Mechanisms
    Article Snippet: The precipitated proteins were pelleted (Beckman JA-14 rotor, 14,000 rpm, 30 min, 4°C), dissolved in 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol, and run through a 10-ml amylose column (FPLC; Amylose High-Flow resin; New England BioLabs, Ipswich, MA), which was washed with 3 column volumes of 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol and eluted with 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol containing 10 mM maltose. .. Fractions containing the MalE-LtrA fusion were incubated with TEV protease (80 µg/ml, 18 h, at 4°C), and imidazole was added to a final concentration of 40 mM.

    Article Title: Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers
    Article Snippet: Briefly, 18–35 nt mouse small RNA was purified from a 15% denaturing urea-polyacrylamide gel (National Diagnostics, Atlanta, GA, USA). .. Small RNA was first ligated to 3′ DNA adapters with adenylated 5′ and dideoxycytosine-blocked 3′ ends in 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 10 mM DTT, and 50% PEG 8000 (New England Biolabs, Ipswich, MA, USA) with T4 Rnl2tr K227Q (homemade) at 25 °C for 16 h. The 3′ adapter contained UMIs in 3 nt-blocks of random nucleotides separated by pre-defined 3 nt consensus sequences (NNN-GTC-NNN-TAG-NNN, Fig. ).

    Polymerase Chain Reaction:

    Article Title: Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers
    Article Snippet: Small RNA was first ligated to 3′ DNA adapters with adenylated 5′ and dideoxycytosine-blocked 3′ ends in 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 10 mM DTT, and 50% PEG 8000 (New England Biolabs, Ipswich, MA, USA) with T4 Rnl2tr K227Q (homemade) at 25 °C for 16 h. The 3′ adapter contained UMIs in 3 nt-blocks of random nucleotides separated by pre-defined 3 nt consensus sequences (NNN-GTC-NNN-TAG-NNN, Fig. ). .. The 54–71 nt (18–35 nt small RNA + 36 nt 3′ UMI adapter) 3′ ligated product was purified from a 10% denaturing urea-polyacrylamide gel.

    Degradation Assay:

    Article Title: Lysosomal membrane protein SIDT2 mediates the direct uptake of DNA by lysosomes
    Article Snippet: Paragraph title: DNA degradation assay ... Following incubation, DNA was extracted as follows: A proteinase K solution containing 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% SDS, and 0.1 mg/ml proteinase K (New England Biolabs Inc., P8102S) in saline sodium citrate buffer was added and incubated for 2 h at 37°C, followed by phenol-chloroform extraction and ethanol precipitation.

    Fast Protein Liquid Chromatography:

    Article Title: The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4-Dependent and -Independent Mechanisms
    Article Snippet: Proteins were precipitated from the supernatant by adding ammonium sulfate to 50% saturation with constant stirring for 1 h at 4°C. .. The precipitated proteins were pelleted (Beckman JA-14 rotor, 14,000 rpm, 30 min, 4°C), dissolved in 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol, and run through a 10-ml amylose column (FPLC; Amylose High-Flow resin; New England BioLabs, Ipswich, MA), which was washed with 3 column volumes of 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol and eluted with 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol containing 10 mM maltose. .. Fractions containing the MalE-LtrA fusion were incubated with TEV protease (80 µg/ml, 18 h, at 4°C), and imidazole was added to a final concentration of 40 mM.

    De-Phosphorylation Assay:

    Article Title: DISC1-dependent switch from progenitor proliferation to migration in the developing cortex
    Article Snippet: Beads were washed in 20 mM Tris-HCl, pH 7.6, three times and in lambda phosphatase buffer (New England Biolabs) once, and phosphatase reactions were performed directly on the beads at 30°C for 2 h with lambda phosphatase (New England Biolabs) as per manufacturer's protocol. .. Mouse brains were homogenized in lysis buffer.

    Staining:

    Article Title: Cotranslational assembly of protein complexes in eukaryotes revealed by ribosome profiling
    Article Snippet: Samples were run on a 10% TBE-Urea polyacrylamide gel (Invitrogen) in 1x TBE (Ambion) for 50 min at 200 V. Gels were stained for 20 min with SYBR gold and desired gel pieces were excised and RNA was extracted as described before. .. For reverse transcription, RNA was resuspended in 10 µl 10 mM Tris-HCl pH 7.0 and 1 µl 10 mM dNTP (NEB), 1 µl 25 linker L1’L20 and 1.5µl DEPC H20 were added to each sample.

    Article Title: Stress-Induced Low Complexity RNA Activates Physiological Amyloidogenesis
    Article Snippet: Paragraph title: Congo red, ANS and A11 staining ... Post-fixation for 30 min, cells were quenched with 0.1M Tris-HCl, pH 7.0 for 10 min before Proteinase K (PK) treatment (NEB, 800U/ml stock, 100,000× dilution) at 37°C for 30 min. PK treatment was followed by a 5-min 1% formaldehyde post-fix.

    Article Title: Adaptation to DNA damage checkpoint in senescent telomerase-negative cells promotes genome instability
    Article Snippet: The solution was then replaced with 10 mM Tris-HCl (pH 8.0) containing 50 mM EDTA, 1% sarkosyl, and 2 mg/mL proteinase K (New England Biolabs), and the plugs were incubated for 24 h at 50°C. .. After extensive washing with 10 mM Tris-HCl and 50 mM EDTA (pH 8.0), the plugs were loaded into the wells of a 0.9% agarose gel (Seakem Gold, Ozyme) and electrophoresed at 13°C in a rotating PFGE apparatus (Rotaphor 6.0, Biometra) according to the manufacturer's instructions for separation of S. cerevisiae chromosomes.

    cDNA Library Assay:

    Article Title: Cotranslational assembly of protein complexes in eukaryotes revealed by ribosome profiling
    Article Snippet: Paragraph title: cDNA library preparation for deep sequencing ... For reverse transcription, RNA was resuspended in 10 µl 10 mM Tris-HCl pH 7.0 and 1 µl 10 mM dNTP (NEB), 1 µl 25 linker L1’L20 and 1.5µl DEPC H20 were added to each sample.

    Activated Clotting Time Assay:

    Article Title: Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers
    Article Snippet: Small RNA was first ligated to 3′ DNA adapters with adenylated 5′ and dideoxycytosine-blocked 3′ ends in 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 10 mM DTT, and 50% PEG 8000 (New England Biolabs, Ipswich, MA, USA) with T4 Rnl2tr K227Q (homemade) at 25 °C for 16 h. The 3′ adapter contained UMIs in 3 nt-blocks of random nucleotides separated by pre-defined 3 nt consensus sequences (NNN-GTC-NNN-TAG-NNN, Fig. ). .. The 54–71 nt (18–35 nt small RNA + 36 nt 3′ UMI adapter) 3′ ligated product was purified from a 10% denaturing urea-polyacrylamide gel.

    SDS Page:

    Article Title: The role of Rab6a and phosphorylation of non-muscle myosin IIA tailpiece in alcohol-induced Golgi disorganization
    Article Snippet: Phosphorylation assay Cell samples were re-suspended in an EDTA-free CIP buffer, containing 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , and 1 mM DTT (New England Biolabs). .. Phosphorylation assay Cell samples were re-suspended in an EDTA-free CIP buffer, containing 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , and 1 mM DTT (New England Biolabs).

    Article Title: Restoration of Compact Golgi Morphology in Advanced Prostate Cancer Enhances Susceptibility to Galectin-1-induced Apoptosis by Modifying Mucin O-glycan Synthesis
    Article Snippet: Cell samples were resuspended in EDTA-free CIP buffer, containing 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , and 1 mM DTT (New England Biolabs). .. Cell samples were resuspended in EDTA-free CIP buffer, containing 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , and 1 mM DTT (New England Biolabs).

    Plasmid Preparation:

    Article Title: The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4-Dependent and -Independent Mechanisms
    Article Snippet: The LtrA protein used in biochemical assays was expressed in E. coli BL21(DE3) from the plasmid pMAL-LtrA. .. The precipitated proteins were pelleted (Beckman JA-14 rotor, 14,000 rpm, 30 min, 4°C), dissolved in 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol, and run through a 10-ml amylose column (FPLC; Amylose High-Flow resin; New England BioLabs, Ipswich, MA), which was washed with 3 column volumes of 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol and eluted with 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol containing 10 mM maltose.

    Article Title: Lysosomal membrane protein SIDT2 mediates the direct uptake of DNA by lysosomes
    Article Snippet: Intact isolated lysosomes were incubated with plasmid DNA in the presence of an ATP regeneration system for 5 min at 37°C. .. Following incubation, DNA was extracted as follows: A proteinase K solution containing 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% SDS, and 0.1 mg/ml proteinase K (New England Biolabs Inc., P8102S) in saline sodium citrate buffer was added and incubated for 2 h at 37°C, followed by phenol-chloroform extraction and ethanol precipitation.

    Software:

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: OGG1 proteins or nuclear extracts were incubated with DNA substrates at varying concentrations in 20 µl reactions containing 20 mM Tris–HCl (pH 7.4), 100 mM NaCl and 0.15 µg/µl BSA (New England Biolabs). .. When measuring AP-lyase activity with an AP site substrate, reactions were terminated by adding SDS and glycerol to 5 and 10%, respectively, without heating.

    Article Title: Lysosomal membrane protein SIDT2 mediates the direct uptake of DNA by lysosomes
    Article Snippet: Following incubation, DNA was extracted as follows: A proteinase K solution containing 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% SDS, and 0.1 mg/ml proteinase K (New England Biolabs Inc., P8102S) in saline sodium citrate buffer was added and incubated for 2 h at 37°C, followed by phenol-chloroform extraction and ethanol precipitation. .. Following incubation, DNA was extracted as follows: A proteinase K solution containing 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% SDS, and 0.1 mg/ml proteinase K (New England Biolabs Inc., P8102S) in saline sodium citrate buffer was added and incubated for 2 h at 37°C, followed by phenol-chloroform extraction and ethanol precipitation.

    SYBR Green Assay:

    Article Title: A Phenolic Fraction from Catharanthus roseus L. Stems Decreases Glycemia and Stimulates Insulin Secretion
    Article Snippet: The reaction was carried out with 1 μ g of RNA in a final volume of 20 μ L. The reaction mixture contained 4 μ M hexamers (Fermentas™, UK), 1X Reverse Transcriptase Buffer 75 mM KCl, 50 mM Tris-HCl, 3 mM MgCl2, 10 mM dithiothreitol (New-England Biolabs™, USA), 10 μ M dNTPs (Promega®, USA), 20 U RNase Inhibitor (Ambion™, USA), and 100 U MuLV Reverse Transcriptase (New-England Biolabs™, USA). .. The reaction was carried out with 1 μ g of RNA in a final volume of 20 μ L. The reaction mixture contained 4 μ M hexamers (Fermentas™, UK), 1X Reverse Transcriptase Buffer 75 mM KCl, 50 mM Tris-HCl, 3 mM MgCl2, 10 mM dithiothreitol (New-England Biolabs™, USA), 10 μ M dNTPs (Promega®, USA), 20 U RNase Inhibitor (Ambion™, USA), and 100 U MuLV Reverse Transcriptase (New-England Biolabs™, USA).

    RNA Extraction:

    Article Title: A Phenolic Fraction from Catharanthus roseus L. Stems Decreases Glycemia and Stimulates Insulin Secretion
    Article Snippet: RNA extraction was performed with Trizol™ following the manufacturer's instructions. .. The reaction was carried out with 1 μ g of RNA in a final volume of 20 μ L. The reaction mixture contained 4 μ M hexamers (Fermentas™, UK), 1X Reverse Transcriptase Buffer 75 mM KCl, 50 mM Tris-HCl, 3 mM MgCl2, 10 mM dithiothreitol (New-England Biolabs™, USA), 10 μ M dNTPs (Promega®, USA), 20 U RNase Inhibitor (Ambion™, USA), and 100 U MuLV Reverse Transcriptase (New-England Biolabs™, USA).

    Selection:

    Article Title: Selection of Single-Stranded DNA Molecular Recognition Elements against Exotoxin A Using a Novel Decoy-SELEX Method and Sensitive Detection of Exotoxin A in Human Serum
    Article Snippet: Paragraph title: 2.1. Decoy-SELEX Method for Selection of Exotoxin A Specific MREs ... Immobilization substrate was prepared by incubating Sulfo-NHS-LC-Biotin (Pierce; Rockford, IL, USA) with Tris-HCl and streptavidin-coated magnetic beads (New England Biolabs, Ipswich, MA, USA).

    Agarose Gel Electrophoresis:

    Article Title: Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers
    Article Snippet: Small RNA was first ligated to 3′ DNA adapters with adenylated 5′ and dideoxycytosine-blocked 3′ ends in 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 10 mM DTT, and 50% PEG 8000 (New England Biolabs, Ipswich, MA, USA) with T4 Rnl2tr K227Q (homemade) at 25 °C for 16 h. The 3′ adapter contained UMIs in 3 nt-blocks of random nucleotides separated by pre-defined 3 nt consensus sequences (NNN-GTC-NNN-TAG-NNN, Fig. ). .. The 3′ ligated product was then ligated to a mixed pool of equimolar amount of 5′ RNA adapters containing UMIs in 3 nt-blocks of random nucleotides and one of the two distinct consensus sequence sets (NNN-CGA-NNN-UAC-NNN and NNN-AUC-NNN-AGU-NNN) in 50 mM Tris-HCl (pH 7.8), 10 mM MgCl2 , 10 mM DTT, 1 mM ATP with T4 RNA ligase (Ambion, Foster City, CA, USA) at 25 °C for 2 h. The ligated product was precipitated with ethanol, and cDNA synthesis was performed using AMV reverse transcriptase (New England Biolabs, Ipswich, MA, USA). cDNA was PCR-amplified with a common forward primer (5′–AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GA-3′) and a reverse primer containing 6 nt Illumina multiplexing barcode (5′–CAA GCA GAA GAC GGC ATA CGA GAT NNN NNN GTG ACT GGA GTT CCT TGG CAC CCG AGA ATT CCA–3′) using AccuPrime Pfx DNA polymerase (ThermoFisher, Waltham, MA, USA).

    Article Title: A Phenolic Fraction from Catharanthus roseus L. Stems Decreases Glycemia and Stimulates Insulin Secretion
    Article Snippet: The concentration and purity of the RNA were determined by spectrophotometry (EPOCH) at A260 and A280 nm, with an A260/A280 ratio of 1.8-2.2 indicating pure RNA, while the RNA integrity was analyzed by agarose gel electrophoresis. .. The reaction was carried out with 1 μ g of RNA in a final volume of 20 μ L. The reaction mixture contained 4 μ M hexamers (Fermentas™, UK), 1X Reverse Transcriptase Buffer 75 mM KCl, 50 mM Tris-HCl, 3 mM MgCl2, 10 mM dithiothreitol (New-England Biolabs™, USA), 10 μ M dNTPs (Promega®, USA), 20 U RNase Inhibitor (Ambion™, USA), and 100 U MuLV Reverse Transcriptase (New-England Biolabs™, USA).

    Ethanol Precipitation:

    Article Title: Lysosomal membrane protein SIDT2 mediates the direct uptake of DNA by lysosomes
    Article Snippet: Intact isolated lysosomes were incubated with plasmid DNA in the presence of an ATP regeneration system for 5 min at 37°C. .. Following incubation, DNA was extracted as follows: A proteinase K solution containing 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% SDS, and 0.1 mg/ml proteinase K (New England Biolabs Inc., P8102S) in saline sodium citrate buffer was added and incubated for 2 h at 37°C, followed by phenol-chloroform extraction and ethanol precipitation. .. Total levels of DNA were analyzed by agarose gel electrophoresis with ethidium bromide staining and UV illumination.

    Article Title: Selection of Single-Stranded DNA Molecular Recognition Elements against Exotoxin A Using a Novel Decoy-SELEX Method and Sensitive Detection of Exotoxin A in Human Serum
    Article Snippet: Subsequently, 0.1 volumes of 3 M sodium acetate at pH 5.2, 2.5 volumes of cold 100% ethanol, and 10 μ g/mL of glycogen were added to the eluted ssDNA for ethanol precipitation at −80°C. .. Immobilization substrate was prepared by incubating Sulfo-NHS-LC-Biotin (Pierce; Rockford, IL, USA) with Tris-HCl and streptavidin-coated magnetic beads (New England Biolabs, Ipswich, MA, USA).

    Spectrophotometry:

    Article Title: A Phenolic Fraction from Catharanthus roseus L. Stems Decreases Glycemia and Stimulates Insulin Secretion
    Article Snippet: The concentration and purity of the RNA were determined by spectrophotometry (EPOCH) at A260 and A280 nm, with an A260/A280 ratio of 1.8-2.2 indicating pure RNA, while the RNA integrity was analyzed by agarose gel electrophoresis. .. The reaction was carried out with 1 μ g of RNA in a final volume of 20 μ L. The reaction mixture contained 4 μ M hexamers (Fermentas™, UK), 1X Reverse Transcriptase Buffer 75 mM KCl, 50 mM Tris-HCl, 3 mM MgCl2, 10 mM dithiothreitol (New-England Biolabs™, USA), 10 μ M dNTPs (Promega®, USA), 20 U RNase Inhibitor (Ambion™, USA), and 100 U MuLV Reverse Transcriptase (New-England Biolabs™, USA).

    Irradiation:

    Article Title: MDP: A Deinococcus Mn2+-Decapeptide Complex Protects Mice from Ionizing Radiation
    Article Snippet: Typically, 50 μl of the T4 DNA ligase mixtures were irradiated with 60 Co at 12 kGy/h (Shepherd and Associates model 109–68 irradiator) aerobically on ice. .. Following irradiation, 5 μl of each IR-treated T4 DNA ligase sample were assayed for residual ligase activity in separate reaction mixtures (final volume, 50 μl) containing 300 ng of Xba I-linearized pUC19 DNA, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 10 mM dithiothreitol and 1 mM ATP (New England Biolabs). .. T4 DNA ligase/pUC19 DNA mixtures were incubated for 16 h at 16°C, followed by agarose (1%) gel electrophoresis.

    Produced:

    Article Title: Stress-Induced Low Complexity RNA Activates Physiological Amyloidogenesis
    Article Snippet: For the graphical representation of the RNA-seq data, base coverage profiles of GL000220.1, an unplaced contig containing an entire ribosomal cassette, were produced in Integrative Genomics Viewer. .. Post-fixation of 30’, cells were quenched with 0.1M Tris-HCl, pH 7.0 for 10’ before ± Proteinase K (PK) treatment (NEB, 800U/ml stock, 100,000× dilution) at 37°C for 30’.

    Concentration Assay:

    Article Title: A Phenolic Fraction from Catharanthus roseus L. Stems Decreases Glycemia and Stimulates Insulin Secretion
    Article Snippet: The concentration and purity of the RNA were determined by spectrophotometry (EPOCH) at A260 and A280 nm, with an A260/A280 ratio of 1.8-2.2 indicating pure RNA, while the RNA integrity was analyzed by agarose gel electrophoresis. .. The reaction was carried out with 1 μ g of RNA in a final volume of 20 μ L. The reaction mixture contained 4 μ M hexamers (Fermentas™, UK), 1X Reverse Transcriptase Buffer 75 mM KCl, 50 mM Tris-HCl, 3 mM MgCl2, 10 mM dithiothreitol (New-England Biolabs™, USA), 10 μ M dNTPs (Promega®, USA), 20 U RNase Inhibitor (Ambion™, USA), and 100 U MuLV Reverse Transcriptase (New-England Biolabs™, USA).

    Migration:

    Article Title: Lysosomal membrane protein SIDT2 mediates the direct uptake of DNA by lysosomes
    Article Snippet: Following incubation, DNA was extracted as follows: A proteinase K solution containing 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% SDS, and 0.1 mg/ml proteinase K (New England Biolabs Inc., P8102S) in saline sodium citrate buffer was added and incubated for 2 h at 37°C, followed by phenol-chloroform extraction and ethanol precipitation. .. Total levels of DNA were analyzed by agarose gel electrophoresis with ethidium bromide staining and UV illumination.

    Phosphorylation Assay:

    Article Title: The role of Rab6a and phosphorylation of non-muscle myosin IIA tailpiece in alcohol-induced Golgi disorganization
    Article Snippet: Transient transfection of VA-13 cells was carried out using the Lipofectamine 2000 (Life Science technologies) following the manufacturer’s protocol. .. Phosphorylation assay Cell samples were re-suspended in an EDTA-free CIP buffer, containing 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , and 1 mM DTT (New England Biolabs). .. Samples were further treated with CIP (New England Biolabs) (1 unit CIP per microgram of protein) in the absence or presence of a phosphatase inhibitor, β-glycerophosphate (50 mM) for 60 min at 37 °C.

    Lysis:

    Article Title: DISC1-dependent switch from progenitor proliferation to migration in the developing cortex
    Article Snippet: Beads were washed in 20 mM Tris-HCl, pH 7.6, three times and in lambda phosphatase buffer (New England Biolabs) once, and phosphatase reactions were performed directly on the beads at 30°C for 2 h with lambda phosphatase (New England Biolabs) as per manufacturer's protocol. .. Immune complexes were then washed three times in lysis buffer, separated on SDS-PAGE, and analyzed by Western blotting.

    Article Title: MiRAR—miRNA Activity Reporter for Living Cells
    Article Snippet: Briefly, human embryonic kidney 293 (HEK 293) cells were grown to confluency in Dulbecco’s modified eagle medium (DMEM) from Gibco (Thermo Fisher Scientific, Waltham, MA, USA) and 10% fetal bovine serum (FBS). .. Cells from half of a 150 mm plate (~107 cells) were resuspended in 2.4 mL lysis buffer (0.6% sodium dodecyl sulfate (SDS), 10 mM ethylenediaminetetraacetic acid (EDTA), 10 mM Tris-HCl pH 8.0) and 25 units RNase I (NEB M0243S) and incubated at 37 °C for 1 h. This was followed by the addition of 240 µL 5 M NaCl (6 mmol) and 1 h of incubation on ice. .. The solution was centrifuged at 10,000× g for 30 min at 4 °C and the DNA was extracted from the supernatant via phenol chloroform extraction.

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  • 99
    New England Biolabs bamhi buffer
    Variant M91V/E156K digestion of plasmid substrate pUC-GCT. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at <t>37°C</t> for 60 min in 1 × NEB <t>BamHI</t> buffer. (−) indicates no enzyme addition.
    Bamhi Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bamhi buffer/product/New England Biolabs
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    bamhi buffer - by Bioz Stars, 2019-10
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    82
    New England Biolabs l30 binding buffer
    REMSA analysis of the RNA-binding activity of <t>L30.</t> A . The 32 P-labeled PHGPx SECIS was incubated with increasing amounts of L30 as indicated. The complexes were analyzed by native gel electrophoresis (top panel). The percent SECIS bound at each protein concentration is shown graphically (bottom panel). B . REMSA analysis was performed as described in (A) except that the stem-loop from the L30 pre-mRNA (L30 RNA) was used as the probe.
    L30 Binding Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 82/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs buffer 3
    Effects of exogenous phosphatase and RNase treatment on capsid assembly in RRL. The indicated HBc proteins were translated in RRL, and the translation reaction mixtures were resolved by agarose gel electrophoresis (top panels) or SDS-PAGE (bottom panels) without any further treatment (lanes 1, 7, 13, 19, 25, and 31) or were treated with NEB <t>buffer</t> 3 alone overnight at 37°C (buffer) (lanes 2, 8, 14, 20, 26, and 32), with buffer 3 plus CIAP overnight at 37°C (CIAP) (lanes 3, 9, 15, 21, 27, and 33), with buffer 3 plus CIAP overnight at 37°C followed by RNase treatment for one additional hour (CIAP-RNase) (lanes 4, 10, 16, 22, 28, and 34), with RNase for 1 h followed by buffer 3 plus CIAP overnight at 37°C (lanes 5, 11, 17, 23, 29, and 35), or with the mixture of phosphatase inhibitors overnight at 37°C (lanes 6, 12, 18, 24, 30, and 36). All lanes contained 2 μl translation products. 35 S-labeled HBc proteins were detected by autoradiography. C, 3A, and 3E/7A, WT or mutant HBc subunits; C149, C-terminally truncated HBc protein (terminated at position 149); C-deP, dephosphorylated HBc subunits; Ca, capsids.
    Buffer 3, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Variant M91V/E156K digestion of plasmid substrate pUC-GCT. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1 × NEB BamHI buffer. (−) indicates no enzyme addition.

    Journal: Nucleic Acids Research

    Article Title: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

    doi: 10.1093/nar/gkj483

    Figure Lengend Snippet: Variant M91V/E156K digestion of plasmid substrate pUC-GCT. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1 × NEB BamHI buffer. (−) indicates no enzyme addition.

    Article Snippet: This digestion and all subsequent reactions were carried out at 37°C in NEB BamHI buffer [150 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 1 mM DTT (pH 7.9 at 25°C)].

    Techniques: Variant Assay, Plasmid Preparation, Incubation

    Variant E156K digestion of a substrate containing a single 5′-GCTGCCGC-3′ site. Plasmid pUC-GCT was derived from pUC19. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer.

    Journal: Nucleic Acids Research

    Article Title: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

    doi: 10.1093/nar/gkj483

    Figure Lengend Snippet: Variant E156K digestion of a substrate containing a single 5′-GCTGCCGC-3′ site. Plasmid pUC-GCT was derived from pUC19. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer.

    Article Snippet: This digestion and all subsequent reactions were carried out at 37°C in NEB BamHI buffer [150 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 1 mM DTT (pH 7.9 at 25°C)].

    Techniques: Variant Assay, Plasmid Preparation, Derivative Assay, Incubation

    Digestion of T7 genomic DNA by variant M91V/E156K. ( A ) A virtual digest of T7 DNA using NEBcutter ( 29 ). Since site 5′-GCGTCCGC-3′ is nicked by this enzyme it was not included in the virtual digest. M is a size marker lane. ( B ) Actual results of digesting T7 DNA in 1× NEB BamHI buffer for various times at 37°C. A 30-fold molar excess of enzyme was used in each reaction. ( C ) A control showing no digestion of T7 DNA by 200 U wt NotI.

    Journal: Nucleic Acids Research

    Article Title: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

    doi: 10.1093/nar/gkj483

    Figure Lengend Snippet: Digestion of T7 genomic DNA by variant M91V/E156K. ( A ) A virtual digest of T7 DNA using NEBcutter ( 29 ). Since site 5′-GCGTCCGC-3′ is nicked by this enzyme it was not included in the virtual digest. M is a size marker lane. ( B ) Actual results of digesting T7 DNA in 1× NEB BamHI buffer for various times at 37°C. A 30-fold molar excess of enzyme was used in each reaction. ( C ) A control showing no digestion of T7 DNA by 200 U wt NotI.

    Article Snippet: This digestion and all subsequent reactions were carried out at 37°C in NEB BamHI buffer [150 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 1 mM DTT (pH 7.9 at 25°C)].

    Techniques: Variant Assay, Marker

    Competitive cleavage of two linear substrates by variant E156K/M91V. The 2686 bp substrate was derived from pUC-GCT and the 1778 bp substrate was derived from pUC-NotI. Equimolar amounts of each substrate were mixed and incubated with increasing concentrations of enzyme. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer. (−) indicates no enzyme addition.

    Journal: Nucleic Acids Research

    Article Title: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

    doi: 10.1093/nar/gkj483

    Figure Lengend Snippet: Competitive cleavage of two linear substrates by variant E156K/M91V. The 2686 bp substrate was derived from pUC-GCT and the 1778 bp substrate was derived from pUC-NotI. Equimolar amounts of each substrate were mixed and incubated with increasing concentrations of enzyme. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer. (−) indicates no enzyme addition.

    Article Snippet: This digestion and all subsequent reactions were carried out at 37°C in NEB BamHI buffer [150 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 1 mM DTT (pH 7.9 at 25°C)].

    Techniques: Variant Assay, Derivative Assay, Incubation

    REMSA analysis of the RNA-binding activity of L30. A . The 32 P-labeled PHGPx SECIS was incubated with increasing amounts of L30 as indicated. The complexes were analyzed by native gel electrophoresis (top panel). The percent SECIS bound at each protein concentration is shown graphically (bottom panel). B . REMSA analysis was performed as described in (A) except that the stem-loop from the L30 pre-mRNA (L30 RNA) was used as the probe.

    Journal: BMC Molecular Biology

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element

    doi: 10.1186/1471-2199-14-12

    Figure Lengend Snippet: REMSA analysis of the RNA-binding activity of L30. A . The 32 P-labeled PHGPx SECIS was incubated with increasing amounts of L30 as indicated. The complexes were analyzed by native gel electrophoresis (top panel). The percent SECIS bound at each protein concentration is shown graphically (bottom panel). B . REMSA analysis was performed as described in (A) except that the stem-loop from the L30 pre-mRNA (L30 RNA) was used as the probe.

    Article Snippet: The RNA (2.5 nM) was incubated in L30 binding buffer (30 mM Tris HCl, pH 8.0, 75 mM KCl, 5 mM MgCl2 , 1 mM DTT, 0.04 μg/μL BSA (NEB), 10% glycerol, and 50 ng/μL yeast tRNA) with or without rat L30 protein (0.75 or 1.5 μM as indicated) at 30°C for 15 min.

    Techniques: RNA Binding Assay, Activity Assay, Labeling, Incubation, Nucleic Acid Electrophoresis, Protein Concentration

    L30 repression of UGA recoding correlates with SECIS-binding. UGA recoding assays were performed using the luc/UGA/TR1 reporter construct as described in the legend to Figure 4 A. Reactions contained either no L30 or 44 pmol of wild-type or mutant L30 proteins. Results are expressed as means ± SEM.

    Journal: BMC Molecular Biology

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element

    doi: 10.1186/1471-2199-14-12

    Figure Lengend Snippet: L30 repression of UGA recoding correlates with SECIS-binding. UGA recoding assays were performed using the luc/UGA/TR1 reporter construct as described in the legend to Figure 4 A. Reactions contained either no L30 or 44 pmol of wild-type or mutant L30 proteins. Results are expressed as means ± SEM.

    Article Snippet: The RNA (2.5 nM) was incubated in L30 binding buffer (30 mM Tris HCl, pH 8.0, 75 mM KCl, 5 mM MgCl2 , 1 mM DTT, 0.04 μg/μL BSA (NEB), 10% glycerol, and 50 ng/μL yeast tRNA) with or without rat L30 protein (0.75 or 1.5 μM as indicated) at 30°C for 15 min.

    Techniques: Binding Assay, Construct, Mutagenesis

    The U40C mutation abrogates L30 binding. A . Schematic illustrating the binding sites of SBP2 [ 14 ] and L30 (this work) on the SECIS, as determined by RNA footprinting. The position of the U40C point mutation is indicated. B . UV cross-linking experiments were performed using the 32 P-labeled wild-type PHGPx SECIS or the U40C mutant RNA, which were incubated with increasing amounts of purified L30 as indicated. After RNase digestion, the products were analyzed by SDS-PAGE and autoradiography.

    Journal: BMC Molecular Biology

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element

    doi: 10.1186/1471-2199-14-12

    Figure Lengend Snippet: The U40C mutation abrogates L30 binding. A . Schematic illustrating the binding sites of SBP2 [ 14 ] and L30 (this work) on the SECIS, as determined by RNA footprinting. The position of the U40C point mutation is indicated. B . UV cross-linking experiments were performed using the 32 P-labeled wild-type PHGPx SECIS or the U40C mutant RNA, which were incubated with increasing amounts of purified L30 as indicated. After RNase digestion, the products were analyzed by SDS-PAGE and autoradiography.

    Article Snippet: The RNA (2.5 nM) was incubated in L30 binding buffer (30 mM Tris HCl, pH 8.0, 75 mM KCl, 5 mM MgCl2 , 1 mM DTT, 0.04 μg/μL BSA (NEB), 10% glycerol, and 50 ng/μL yeast tRNA) with or without rat L30 protein (0.75 or 1.5 μM as indicated) at 30°C for 15 min.

    Techniques: Mutagenesis, Binding Assay, Footprinting, Labeling, Incubation, Purification, SDS Page, Autoradiography

    Repression of UGA recoding by L30 is rescued by SBP2. A . In vitro UGA recoding assays were performed using a luciferase reporter RNA that contains UGA (top panel) or UGU (bottom panel) at position 258 of the coding region, fused to either the PHGPx or TR1 SECIS element as indicated. Translation assays were performed in the presence of increasing amounts of L30 as indicated. The products were analyzed for luciferase activity using a luminometer, and the results are expressed as means ± SEM. Statistical significance is indicated by ** ( p

    Journal: BMC Molecular Biology

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element

    doi: 10.1186/1471-2199-14-12

    Figure Lengend Snippet: Repression of UGA recoding by L30 is rescued by SBP2. A . In vitro UGA recoding assays were performed using a luciferase reporter RNA that contains UGA (top panel) or UGU (bottom panel) at position 258 of the coding region, fused to either the PHGPx or TR1 SECIS element as indicated. Translation assays were performed in the presence of increasing amounts of L30 as indicated. The products were analyzed for luciferase activity using a luminometer, and the results are expressed as means ± SEM. Statistical significance is indicated by ** ( p

    Article Snippet: The RNA (2.5 nM) was incubated in L30 binding buffer (30 mM Tris HCl, pH 8.0, 75 mM KCl, 5 mM MgCl2 , 1 mM DTT, 0.04 μg/μL BSA (NEB), 10% glycerol, and 50 ng/μL yeast tRNA) with or without rat L30 protein (0.75 or 1.5 μM as indicated) at 30°C for 15 min.

    Techniques: In Vitro, Luciferase, Activity Assay

    Mutational analysis of L30. A . A schematic illustrating the primary sequences and predicted secondary structures of L30 and SBP2. The position numbers refer to the rat protein sequences. The L7Ae conserved RNA-binding domain is underlined and the conserved signature amino acid motifs for L30 and SBP2 are boxed as described in [ 37 ]. Arrows indicate the amino acids in L30 that were mutated to alanine. B . The wild-type and mutant L30 protein were expressed in bacteria, purified and analyzed by SDS-PAGE and Coomassie Blue staining. The molecular weight markers are shown in the left lane of each gel.

    Journal: BMC Molecular Biology

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element

    doi: 10.1186/1471-2199-14-12

    Figure Lengend Snippet: Mutational analysis of L30. A . A schematic illustrating the primary sequences and predicted secondary structures of L30 and SBP2. The position numbers refer to the rat protein sequences. The L7Ae conserved RNA-binding domain is underlined and the conserved signature amino acid motifs for L30 and SBP2 are boxed as described in [ 37 ]. Arrows indicate the amino acids in L30 that were mutated to alanine. B . The wild-type and mutant L30 protein were expressed in bacteria, purified and analyzed by SDS-PAGE and Coomassie Blue staining. The molecular weight markers are shown in the left lane of each gel.

    Article Snippet: The RNA (2.5 nM) was incubated in L30 binding buffer (30 mM Tris HCl, pH 8.0, 75 mM KCl, 5 mM MgCl2 , 1 mM DTT, 0.04 μg/μL BSA (NEB), 10% glycerol, and 50 ng/μL yeast tRNA) with or without rat L30 protein (0.75 or 1.5 μM as indicated) at 30°C for 15 min.

    Techniques: RNA Binding Assay, Mutagenesis, Purification, SDS Page, Staining, Molecular Weight

    Point mutations in L30 affect RNA-binding activity. A . Representative REMSA analysis of the 32 P-labeled PHGPx SECIS (top panel) or L30 RNA (bottom panel). The RNA probes were incubated in the absence or presence of wild-type and mutant L30 proteins as indicated. The positions of the free probes and protein:RNA complexes are indicated. B . Graphical representation of REMSA results for L30 binding to the SECIS (top panel) or L30 RNA (bottom panel) from 5 or 3 independent experiments, respectively. The results are expressed relative to the activity of the wild-type protein, which is expressed as 100%. Statistical significance is shown by asterisks, with * (p

    Journal: BMC Molecular Biology

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element

    doi: 10.1186/1471-2199-14-12

    Figure Lengend Snippet: Point mutations in L30 affect RNA-binding activity. A . Representative REMSA analysis of the 32 P-labeled PHGPx SECIS (top panel) or L30 RNA (bottom panel). The RNA probes were incubated in the absence or presence of wild-type and mutant L30 proteins as indicated. The positions of the free probes and protein:RNA complexes are indicated. B . Graphical representation of REMSA results for L30 binding to the SECIS (top panel) or L30 RNA (bottom panel) from 5 or 3 independent experiments, respectively. The results are expressed relative to the activity of the wild-type protein, which is expressed as 100%. Statistical significance is shown by asterisks, with * (p

    Article Snippet: The RNA (2.5 nM) was incubated in L30 binding buffer (30 mM Tris HCl, pH 8.0, 75 mM KCl, 5 mM MgCl2 , 1 mM DTT, 0.04 μg/μL BSA (NEB), 10% glycerol, and 50 ng/μL yeast tRNA) with or without rat L30 protein (0.75 or 1.5 μM as indicated) at 30°C for 15 min.

    Techniques: RNA Binding Assay, Activity Assay, Labeling, Incubation, Mutagenesis, Binding Assay

    RNA footprinting of the L30:SECIS complex. A . The structure of the SECIS element from the rat PHGPx mRNA is shown. Boxes and circles indicate nucleotides that are protected from cleavage by RNase T1 and RNase A, respectively. B . The 5’ end-labeled PHGPx SECIS was incubated in the absence or presence of L30 (0.75 or 1.5 μM). The reactions were then partially digested with RNase T1, A, or V1 as indicated. The products were analyzed by denaturing gel electrophoresis. The sequencing (G and C + U) and alkali ladders are shown in the left lanes. The numbers to the left of the gel indicate the positions of G nucleotides using the numbering in (A). The bars on the right indicate the different regions of the SECIS element. The gel is a representative example from 3 independent experiments.

    Journal: BMC Molecular Biology

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element

    doi: 10.1186/1471-2199-14-12

    Figure Lengend Snippet: RNA footprinting of the L30:SECIS complex. A . The structure of the SECIS element from the rat PHGPx mRNA is shown. Boxes and circles indicate nucleotides that are protected from cleavage by RNase T1 and RNase A, respectively. B . The 5’ end-labeled PHGPx SECIS was incubated in the absence or presence of L30 (0.75 or 1.5 μM). The reactions were then partially digested with RNase T1, A, or V1 as indicated. The products were analyzed by denaturing gel electrophoresis. The sequencing (G and C + U) and alkali ladders are shown in the left lanes. The numbers to the left of the gel indicate the positions of G nucleotides using the numbering in (A). The bars on the right indicate the different regions of the SECIS element. The gel is a representative example from 3 independent experiments.

    Article Snippet: The RNA (2.5 nM) was incubated in L30 binding buffer (30 mM Tris HCl, pH 8.0, 75 mM KCl, 5 mM MgCl2 , 1 mM DTT, 0.04 μg/μL BSA (NEB), 10% glycerol, and 50 ng/μL yeast tRNA) with or without rat L30 protein (0.75 or 1.5 μM as indicated) at 30°C for 15 min.

    Techniques: Footprinting, Labeling, Incubation, Nucleic Acid Electrophoresis, Sequencing

    Effects of exogenous phosphatase and RNase treatment on capsid assembly in RRL. The indicated HBc proteins were translated in RRL, and the translation reaction mixtures were resolved by agarose gel electrophoresis (top panels) or SDS-PAGE (bottom panels) without any further treatment (lanes 1, 7, 13, 19, 25, and 31) or were treated with NEB buffer 3 alone overnight at 37°C (buffer) (lanes 2, 8, 14, 20, 26, and 32), with buffer 3 plus CIAP overnight at 37°C (CIAP) (lanes 3, 9, 15, 21, 27, and 33), with buffer 3 plus CIAP overnight at 37°C followed by RNase treatment for one additional hour (CIAP-RNase) (lanes 4, 10, 16, 22, 28, and 34), with RNase for 1 h followed by buffer 3 plus CIAP overnight at 37°C (lanes 5, 11, 17, 23, 29, and 35), or with the mixture of phosphatase inhibitors overnight at 37°C (lanes 6, 12, 18, 24, 30, and 36). All lanes contained 2 μl translation products. 35 S-labeled HBc proteins were detected by autoradiography. C, 3A, and 3E/7A, WT or mutant HBc subunits; C149, C-terminally truncated HBc protein (terminated at position 149); C-deP, dephosphorylated HBc subunits; Ca, capsids.

    Journal:

    Article Title: Cell-Free Hepatitis B Virus Capsid Assembly Dependent on the Core Protein C-Terminal Domain and Regulated by Phosphorylation

    doi: 10.1128/JVI.00394-16

    Figure Lengend Snippet: Effects of exogenous phosphatase and RNase treatment on capsid assembly in RRL. The indicated HBc proteins were translated in RRL, and the translation reaction mixtures were resolved by agarose gel electrophoresis (top panels) or SDS-PAGE (bottom panels) without any further treatment (lanes 1, 7, 13, 19, 25, and 31) or were treated with NEB buffer 3 alone overnight at 37°C (buffer) (lanes 2, 8, 14, 20, 26, and 32), with buffer 3 plus CIAP overnight at 37°C (CIAP) (lanes 3, 9, 15, 21, 27, and 33), with buffer 3 plus CIAP overnight at 37°C followed by RNase treatment for one additional hour (CIAP-RNase) (lanes 4, 10, 16, 22, 28, and 34), with RNase for 1 h followed by buffer 3 plus CIAP overnight at 37°C (lanes 5, 11, 17, 23, 29, and 35), or with the mixture of phosphatase inhibitors overnight at 37°C (lanes 6, 12, 18, 24, 30, and 36). All lanes contained 2 μl translation products. 35 S-labeled HBc proteins were detected by autoradiography. C, 3A, and 3E/7A, WT or mutant HBc subunits; C149, C-terminally truncated HBc protein (terminated at position 149); C-deP, dephosphorylated HBc subunits; Ca, capsids.

    Article Snippet: Unless specifically indicated otherwise, the general assembly reaction mixtures included 1 to 3 μl of translation products per 10 μl final reaction volume in 1× buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 1 mM dithiothreitol, pH 7.9; New England BioLabs, or NEB) supplemented with 1× EDTA-free protease inhibitor cocktail (Roche) and 0.8 U/μl RNasin Plus RNase inhibitor (Promega).

    Techniques: Agarose Gel Electrophoresis, SDS Page, Labeling, Autoradiography, Mutagenesis

    HBV capsid assembly in RRL and effects of exogenous phosphatase and phosphatase inhibitors on assembly. The WT and mutant HBc proteins or the control luciferase (Luc) was translated in RRL. All samples were resolved by agarose gel electrophoresis. (A) The indicated protein translated in RRL was incubated overnight at 37°C in 1× NEB restriction digestion buffer 3 alone (lanes 1, 3, 5, 7, and 9) or with CIAP (lanes 2, 4, 6, 8, and 10) before resolution on the gel. The recombinant HBV capsid (rHBc) purified from E. coli was loaded in lane 11. (B) The indicated translation reaction mixture was loaded directly following translation upon dilution in double-distilled water (dH2 O) and without the overnight incubation (i.e., no assembly reaction) (lanes 1, 5, 9, and 13), after dilution in NEB buffer 3 (buffer 3) but without the overnight incubation (lanes 2, 6, 10, and 14), after dilution in buffer 3 and with incubation overnight at 37°C (lanes 3, 7, 11, and 15), or after overnight incubation in buffer 3 and with CIAP (lanes 4, 8, 12, and 16). (C) The indicated translation reaction mixture was loaded directly following translation upon dilution in dH2 O and without the overnight incubation (i.e., no assembly reaction) (lanes 1, 5, and 9), after dilution in dH2 O and with incubation overnight at 37°C (lanes 2, 6, and 10), after dilution in buffer 3 and with incubation overnight at 37°C (lanes 3, 7, and 11), or after overnight incubation at 37°C in buffer 3 and with a mixture of phosphatase inhibitors (PPI) (lanes 4, 8, and 12). Each lane contained 3 μl translation product except that 3.125 ng rHBc was loaded in lane 11 of panel A. 35 S signals were detected by autoradiography (top). The HBc proteins were also detected by the MAb antibody against the NTD (bottom). C/3A/3E, WT, 3A, or 3E HBc subunits (i.e., not present in the capsid); C149, C-terminally truncated HBc protein (terminated at position 149); C-deP, dephosphorylated WT HBc subunits; Ca, capsids.

    Journal:

    Article Title: Cell-Free Hepatitis B Virus Capsid Assembly Dependent on the Core Protein C-Terminal Domain and Regulated by Phosphorylation

    doi: 10.1128/JVI.00394-16

    Figure Lengend Snippet: HBV capsid assembly in RRL and effects of exogenous phosphatase and phosphatase inhibitors on assembly. The WT and mutant HBc proteins or the control luciferase (Luc) was translated in RRL. All samples were resolved by agarose gel electrophoresis. (A) The indicated protein translated in RRL was incubated overnight at 37°C in 1× NEB restriction digestion buffer 3 alone (lanes 1, 3, 5, 7, and 9) or with CIAP (lanes 2, 4, 6, 8, and 10) before resolution on the gel. The recombinant HBV capsid (rHBc) purified from E. coli was loaded in lane 11. (B) The indicated translation reaction mixture was loaded directly following translation upon dilution in double-distilled water (dH2 O) and without the overnight incubation (i.e., no assembly reaction) (lanes 1, 5, 9, and 13), after dilution in NEB buffer 3 (buffer 3) but without the overnight incubation (lanes 2, 6, 10, and 14), after dilution in buffer 3 and with incubation overnight at 37°C (lanes 3, 7, 11, and 15), or after overnight incubation in buffer 3 and with CIAP (lanes 4, 8, 12, and 16). (C) The indicated translation reaction mixture was loaded directly following translation upon dilution in dH2 O and without the overnight incubation (i.e., no assembly reaction) (lanes 1, 5, and 9), after dilution in dH2 O and with incubation overnight at 37°C (lanes 2, 6, and 10), after dilution in buffer 3 and with incubation overnight at 37°C (lanes 3, 7, and 11), or after overnight incubation at 37°C in buffer 3 and with a mixture of phosphatase inhibitors (PPI) (lanes 4, 8, and 12). Each lane contained 3 μl translation product except that 3.125 ng rHBc was loaded in lane 11 of panel A. 35 S signals were detected by autoradiography (top). The HBc proteins were also detected by the MAb antibody against the NTD (bottom). C/3A/3E, WT, 3A, or 3E HBc subunits (i.e., not present in the capsid); C149, C-terminally truncated HBc protein (terminated at position 149); C-deP, dephosphorylated WT HBc subunits; Ca, capsids.

    Article Snippet: Unless specifically indicated otherwise, the general assembly reaction mixtures included 1 to 3 μl of translation products per 10 μl final reaction volume in 1× buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 1 mM dithiothreitol, pH 7.9; New England BioLabs, or NEB) supplemented with 1× EDTA-free protease inhibitor cocktail (Roche) and 0.8 U/μl RNasin Plus RNase inhibitor (Promega).

    Techniques: Mutagenesis, Luciferase, Agarose Gel Electrophoresis, Incubation, Recombinant, Purification, Autoradiography