triton x 100  (New England Biolabs)


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    Structured Review

    New England Biolabs triton x 100
    Pulse-chase analysis of HCMV glycoproteins expressed in cells and incorporated into extracellular virus particles. Human fibroblasts (NHDF) were infected with 3 IU/cell of HCMV TR for 4 days. The cells were labeled for 1 h with [ 35 S]methionine/cysteine (500 uCi/ml), and then the label was chased for 3 or 24 h. The following three fractions were prepared: cells (C), virus particles derived from cell culture supernatants by centrifugation through 20% sorbitol cushions (V), and the cell culture supernatants after viruses were pelleted (S). Each fraction was extracted with 1% Triton X-100, and then gH or gO was immunoprecipitated using anti-gH MAb 14-4b (A) or anti-TR gO254 antibodies (B). Immunoprecipitated proteins were left untreated or treated with endo H. Arrows indicate bands corresponding to gH, gO, gL, or endo H-sensitive forms of these proteins (H). Arrowheads mark unidentified proteins that coprecipitated with gH. Molecular weight markers are shown along the left side of each panel.
    Triton X 100, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Human Cytomegalovirus TR Strain Glycoprotein O Acts as a Chaperone Promoting gH/gL Incorporation into Virions but Is Not Present in Virions ▿"

    Article Title: Human Cytomegalovirus TR Strain Glycoprotein O Acts as a Chaperone Promoting gH/gL Incorporation into Virions but Is Not Present in Virions ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02256-09

    Pulse-chase analysis of HCMV glycoproteins expressed in cells and incorporated into extracellular virus particles. Human fibroblasts (NHDF) were infected with 3 IU/cell of HCMV TR for 4 days. The cells were labeled for 1 h with [ 35 S]methionine/cysteine (500 uCi/ml), and then the label was chased for 3 or 24 h. The following three fractions were prepared: cells (C), virus particles derived from cell culture supernatants by centrifugation through 20% sorbitol cushions (V), and the cell culture supernatants after viruses were pelleted (S). Each fraction was extracted with 1% Triton X-100, and then gH or gO was immunoprecipitated using anti-gH MAb 14-4b (A) or anti-TR gO254 antibodies (B). Immunoprecipitated proteins were left untreated or treated with endo H. Arrows indicate bands corresponding to gH, gO, gL, or endo H-sensitive forms of these proteins (H). Arrowheads mark unidentified proteins that coprecipitated with gH. Molecular weight markers are shown along the left side of each panel.
    Figure Legend Snippet: Pulse-chase analysis of HCMV glycoproteins expressed in cells and incorporated into extracellular virus particles. Human fibroblasts (NHDF) were infected with 3 IU/cell of HCMV TR for 4 days. The cells were labeled for 1 h with [ 35 S]methionine/cysteine (500 uCi/ml), and then the label was chased for 3 or 24 h. The following three fractions were prepared: cells (C), virus particles derived from cell culture supernatants by centrifugation through 20% sorbitol cushions (V), and the cell culture supernatants after viruses were pelleted (S). Each fraction was extracted with 1% Triton X-100, and then gH or gO was immunoprecipitated using anti-gH MAb 14-4b (A) or anti-TR gO254 antibodies (B). Immunoprecipitated proteins were left untreated or treated with endo H. Arrows indicate bands corresponding to gH, gO, gL, or endo H-sensitive forms of these proteins (H). Arrowheads mark unidentified proteins that coprecipitated with gH. Molecular weight markers are shown along the left side of each panel.

    Techniques Used: Pulse Chase, Infection, Labeling, Derivative Assay, Cell Culture, Centrifugation, Immunoprecipitation, Molecular Weight

    Immunoblot analysis of viral glycoproteins in HCMV TR- and AD169-infected cells and extracellular virus particles. Human fibroblasts (NHDF) were infected with 2 IU per cell of TR or AD169 for 7 days. Extracellular viral particles were purified from clarified culture supernatants by centrifugation though 20% sorbitol cushions (V). Virus particles and infected cells (C) were extracted with 1% Triton X-100 and analyzed by Western blotting using mouse MAb specific for gB (27-156) or calnexin or rabbit polyclonal antibodies specific for gH, gL, TR gO (TR gO254 produced against gO residues 254 to 271), AD169 gO, or UL130. Note that the TR gO254 antibodies do not recognize AD169 gO, and AD169 gO antibodies do not recognize TR gO; thus, separate blots are shown. A longer exposure of the TR gO blot was provided to show the very small quantity of TR gO in virus particles. Numbers below each lane indicate the relative abundance of each protein. Shown are representative results from three independent experiments. Molecular weight markers are shown along the left side of each panel.
    Figure Legend Snippet: Immunoblot analysis of viral glycoproteins in HCMV TR- and AD169-infected cells and extracellular virus particles. Human fibroblasts (NHDF) were infected with 2 IU per cell of TR or AD169 for 7 days. Extracellular viral particles were purified from clarified culture supernatants by centrifugation though 20% sorbitol cushions (V). Virus particles and infected cells (C) were extracted with 1% Triton X-100 and analyzed by Western blotting using mouse MAb specific for gB (27-156) or calnexin or rabbit polyclonal antibodies specific for gH, gL, TR gO (TR gO254 produced against gO residues 254 to 271), AD169 gO, or UL130. Note that the TR gO254 antibodies do not recognize AD169 gO, and AD169 gO antibodies do not recognize TR gO; thus, separate blots are shown. A longer exposure of the TR gO blot was provided to show the very small quantity of TR gO in virus particles. Numbers below each lane indicate the relative abundance of each protein. Shown are representative results from three independent experiments. Molecular weight markers are shown along the left side of each panel.

    Techniques Used: Infection, Purification, Centrifugation, Western Blot, Produced, Molecular Weight

    Immunoblot analysis of the maturation of HCMV glycoproteins in virions and cells. TR- and AD169-infected fibroblast extracts and extracts of extracellular particles were prepared using 1% Triton X-100. Extracts were either left untreated or treated with endo H or PNGase F, and then proteins were subjected to gel electrophoresis and immunoblot analysis using rabbit polyclonal antibodies specific for gH, gL, or gO. (A) TR gO254 were used to detect gO; (B) AD169 gO-specific antibodies were used. Molecular weight markers are shown along the left side of each panel.
    Figure Legend Snippet: Immunoblot analysis of the maturation of HCMV glycoproteins in virions and cells. TR- and AD169-infected fibroblast extracts and extracts of extracellular particles were prepared using 1% Triton X-100. Extracts were either left untreated or treated with endo H or PNGase F, and then proteins were subjected to gel electrophoresis and immunoblot analysis using rabbit polyclonal antibodies specific for gH, gL, or gO. (A) TR gO254 were used to detect gO; (B) AD169 gO-specific antibodies were used. Molecular weight markers are shown along the left side of each panel.

    Techniques Used: Infection, Nucleic Acid Electrophoresis, Molecular Weight

    2) Product Images from "Erythrocyte membrane-coated nanogel for combinatorial antivirulence and responsive antimicrobial delivery against Staphylococcus aureus infection"

    Article Title: Erythrocyte membrane-coated nanogel for combinatorial antivirulence and responsive antimicrobial delivery against Staphylococcus aureus infection

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    doi: 10.1016/j.jconrel.2017.01.016

    The formulation and characterization of RBC-nanogels. (A) RBC-nanogels loaded with rhodamine B were formulated and subjected to (i) no treatment, (ii) treated with Triton X-100 and proteinase K, or (iii) Triton X-100 and proteinase K followed by tris(2-carboxyethyl) phosphine (TCEP). The RBC-nanogels were then filtered to collect the released dye, which was further measured by a UV-vis spectrophotometer. (B) A representative TEM image of RBC-nanogels (scale bar = 100 nm). (C) Dynamic light scattering (DLS) measurements of the size and size distribution of RBC-vesicles, RBC-nanogels, and non-responsive RBC nanogels (Control nanogels) subjected to the same treatment as in (A).
    Figure Legend Snippet: The formulation and characterization of RBC-nanogels. (A) RBC-nanogels loaded with rhodamine B were formulated and subjected to (i) no treatment, (ii) treated with Triton X-100 and proteinase K, or (iii) Triton X-100 and proteinase K followed by tris(2-carboxyethyl) phosphine (TCEP). The RBC-nanogels were then filtered to collect the released dye, which was further measured by a UV-vis spectrophotometer. (B) A representative TEM image of RBC-nanogels (scale bar = 100 nm). (C) Dynamic light scattering (DLS) measurements of the size and size distribution of RBC-vesicles, RBC-nanogels, and non-responsive RBC nanogels (Control nanogels) subjected to the same treatment as in (A).

    Techniques Used: Spectrophotometry, Transmission Electron Microscopy

    The cumulative release profiles of vancomycin from (A) RBC-nanogels and (B) non-responsive RBC-nanogels (Control nanogels). The nanogels were treated with Triton X-100, treated with Triton X-100 followed by TCEP, or not treated by anything.
    Figure Legend Snippet: The cumulative release profiles of vancomycin from (A) RBC-nanogels and (B) non-responsive RBC-nanogels (Control nanogels). The nanogels were treated with Triton X-100, treated with Triton X-100 followed by TCEP, or not treated by anything.

    Techniques Used:

    3) Product Images from "Lipid Raft-dependent Glucagon-like Peptide-2 Receptor Trafficking Occurs Independently of Agonist-induced Desensitization"

    Article Title: Lipid Raft-dependent Glucagon-like Peptide-2 Receptor Trafficking Occurs Independently of Agonist-induced Desensitization

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E03-11-0825

    A pool of the hGLP-2R is associated with the cellular detergent insoluble fraction and transiently colocalizes with caveolin-1 in transfected cells. (A) DLD-1 cells transiently transfected with FLAG-hGLP-2R or pcDNA3.1 were lysed in a 1% Triton X-100 containing buffer as described in MATERIALS AND METHODS. After centrifugation, the supernatant (Triton X-100–soluble fraction) was recovered, and the pellet was solubilized in the same buffer also containing 0.5% SDS (Triton X-100–insoluble fraction). Aliquots of the Triton X-100–soluble and –insoluble fractions were deglycosylated, and analyzed by immunoblotting for EEA1, hGLP-2R, and caveolin-1. Antigen/antibody complexes were visualized by enhanced chemiluminescence after incubation with a horseradish peroxidase-conjugated secondary antibody. (B) BHK cells transiently expressing the FLAG-hGLP-2R were prelabeled on ice with anti-FLAG antibody and then incubated with media alone (control) or 10 nM hGLP-2R at 37°C for 0–60 min as described in MATERIALS AND METHODS. After fixation and permeabilization, cells were incubated with anti-caveolin-1-FITC. A Cy3-conjugated secondary antibody was used to visualize hGLP-2R/anti-FLAG antibody complexes. Confocal microscopy images are representative of two to three independent experiments.
    Figure Legend Snippet: A pool of the hGLP-2R is associated with the cellular detergent insoluble fraction and transiently colocalizes with caveolin-1 in transfected cells. (A) DLD-1 cells transiently transfected with FLAG-hGLP-2R or pcDNA3.1 were lysed in a 1% Triton X-100 containing buffer as described in MATERIALS AND METHODS. After centrifugation, the supernatant (Triton X-100–soluble fraction) was recovered, and the pellet was solubilized in the same buffer also containing 0.5% SDS (Triton X-100–insoluble fraction). Aliquots of the Triton X-100–soluble and –insoluble fractions were deglycosylated, and analyzed by immunoblotting for EEA1, hGLP-2R, and caveolin-1. Antigen/antibody complexes were visualized by enhanced chemiluminescence after incubation with a horseradish peroxidase-conjugated secondary antibody. (B) BHK cells transiently expressing the FLAG-hGLP-2R were prelabeled on ice with anti-FLAG antibody and then incubated with media alone (control) or 10 nM hGLP-2R at 37°C for 0–60 min as described in MATERIALS AND METHODS. After fixation and permeabilization, cells were incubated with anti-caveolin-1-FITC. A Cy3-conjugated secondary antibody was used to visualize hGLP-2R/anti-FLAG antibody complexes. Confocal microscopy images are representative of two to three independent experiments.

    Techniques Used: Transfection, Centrifugation, Incubation, Expressing, Confocal Microscopy

    Rapid agonist-induced hGLP-2R internalization is followed by slow cell surface receptor reappearance. BHK (A) or DLD-1 (B and C) cells transiently transfected with FLAG-hGLP-2R were treated with vehicle (control), 3 nM, or 10 nM hGLP-2 for either up to 20 min (A and B) or 2 h (C) to assess hGLP-2R internalization. After the hGLP-2 treatment, cells were washed briefly to remove the agonist and incubated for the indicated times in media with serum to assess hGLP-2R reappearance on the cell surface (left). Cell surface receptor levels were measured using an enzyme-linked immunoassay as described in MATERIALS AND METHODS. Data (means ± SD of triplicate values) are expressed as percentage of the cell surface receptor level at time 0. Similar results were obtained in two independent experiments for each cell type. Total cellular levels of FLAG-hGLP-2R in BHK cells (A, right) and DLD-1 cells (B, right) at the end of the internalization (20 min), and washout (200-min) periods were measured as indicated above, after permeabilization of the cells with 0.2% Triton X-100. Data are means ± SD of triplicate values (n = 2 independent experiments for each cell line).
    Figure Legend Snippet: Rapid agonist-induced hGLP-2R internalization is followed by slow cell surface receptor reappearance. BHK (A) or DLD-1 (B and C) cells transiently transfected with FLAG-hGLP-2R were treated with vehicle (control), 3 nM, or 10 nM hGLP-2 for either up to 20 min (A and B) or 2 h (C) to assess hGLP-2R internalization. After the hGLP-2 treatment, cells were washed briefly to remove the agonist and incubated for the indicated times in media with serum to assess hGLP-2R reappearance on the cell surface (left). Cell surface receptor levels were measured using an enzyme-linked immunoassay as described in MATERIALS AND METHODS. Data (means ± SD of triplicate values) are expressed as percentage of the cell surface receptor level at time 0. Similar results were obtained in two independent experiments for each cell type. Total cellular levels of FLAG-hGLP-2R in BHK cells (A, right) and DLD-1 cells (B, right) at the end of the internalization (20 min), and washout (200-min) periods were measured as indicated above, after permeabilization of the cells with 0.2% Triton X-100. Data are means ± SD of triplicate values (n = 2 independent experiments for each cell line).

    Techniques Used: Cell Surface Receptor Assay, Transfection, Incubation

    4) Product Images from "Receptor palmitoylation and ubiquitination regulate anthrax toxin endocytosis"

    Article Title: Receptor palmitoylation and ubiquitination regulate anthrax toxin endocytosis

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200507067

    Palmitoylation events are required for DRM association and internalization of PA.  (A and B) Control BHK cells were pretreated or untreated with bromopalmitate and were incubated with 500 ng/ml nicked PA63 and 20 ng/ml aerolysin for 1 h at 4°C followed by 10 min at 37°C. (A) Cell extracts were submitted to SDS-PAGE followed by Western blotting to reveal PA63, aerolysin, and caveolin-1 (Cav-1). (B) Cells were solubilized in 1% Triton X-100, run on an OptiPrep gradient, and each fraction was analyzed by SDS-PAGE followed by Western blotting against PA, aerolysin, and caveolin-1. (C) BHK cells were pretreated with bromopalmitate and incubated with 500 ng/ml nicked PA63 for 1 h at 4°C followed by different times at 37°C. Cell extracts (40 μg of protein) were analyzed by SDS-PAGE and Western blotting to reveal the SDS-resistant PA 7mer  pore and MEK1 (NH 2 -terminal directed). To detect the prepore (SDS-sensitive nonmembrane-inserted PA 7mer ), cell extracts were submitted to an acid pulse before SDS analysis.
    Figure Legend Snippet: Palmitoylation events are required for DRM association and internalization of PA. (A and B) Control BHK cells were pretreated or untreated with bromopalmitate and were incubated with 500 ng/ml nicked PA63 and 20 ng/ml aerolysin for 1 h at 4°C followed by 10 min at 37°C. (A) Cell extracts were submitted to SDS-PAGE followed by Western blotting to reveal PA63, aerolysin, and caveolin-1 (Cav-1). (B) Cells were solubilized in 1% Triton X-100, run on an OptiPrep gradient, and each fraction was analyzed by SDS-PAGE followed by Western blotting against PA, aerolysin, and caveolin-1. (C) BHK cells were pretreated with bromopalmitate and incubated with 500 ng/ml nicked PA63 for 1 h at 4°C followed by different times at 37°C. Cell extracts (40 μg of protein) were analyzed by SDS-PAGE and Western blotting to reveal the SDS-resistant PA 7mer pore and MEK1 (NH 2 -terminal directed). To detect the prepore (SDS-sensitive nonmembrane-inserted PA 7mer ), cell extracts were submitted to an acid pulse before SDS analysis.

    Techniques Used: Incubation, SDS Page, Western Blot

    Endocytosis of anthrax toxin receptor requires DRM-mediated ubiquitination and the E3 ligase Cbl.  (A) CHO ΔATR  cells transfected for 48 h with WT TEM8/1-HA were incubated with 1 μg/ml PA83 for 1 h at 4°C followed by 40 min at 37°C, solubilized in Triton X-100 at 4°C, and separated on an OptiPrep gradient. TEM8/1-HA was immunoprecipitated from each fraction and analyzed by SDS-PAGE and Western blotting using anti-Ub, anti-HA, and anti-PA antibodies. (B) CHO ΔATR  cells transfected for 48 h with WT TEM8/1-HA were treated with βMCD to extract cholesterol, incubated with 1 μg/ml PA83 for 1 h at 4°C, and shifted for different times at 37°C. After immunoprecipitation with anti-HA beads, samples were analyzed by Western blotting using anti-Ub and anti-HA antibodies. (C) HeLa cells were transfected or untransfected with siRNAs against Cbl for 72 h and incubated with 500 ng/ml PA83 for different times at 37°C. Cell extracts were blotted for Cbl, tubulin (as an equal loading marker), and PA. (D) HeLa cells were untransfected or transfected with siRNAs against Cbl for a total of 72 h in total. 24 h later, these cells were additionally transfected with TEM8/1-HA for 48 h and incubated with 500 ng/ml PA83 for different times at 37°C. TEM8/1-HA was immunoprecipitated from each fraction and analyzed by SDS-PAGE and Western blotting using anti-Ub and anti-HA antibodies.
    Figure Legend Snippet: Endocytosis of anthrax toxin receptor requires DRM-mediated ubiquitination and the E3 ligase Cbl. (A) CHO ΔATR cells transfected for 48 h with WT TEM8/1-HA were incubated with 1 μg/ml PA83 for 1 h at 4°C followed by 40 min at 37°C, solubilized in Triton X-100 at 4°C, and separated on an OptiPrep gradient. TEM8/1-HA was immunoprecipitated from each fraction and analyzed by SDS-PAGE and Western blotting using anti-Ub, anti-HA, and anti-PA antibodies. (B) CHO ΔATR cells transfected for 48 h with WT TEM8/1-HA were treated with βMCD to extract cholesterol, incubated with 1 μg/ml PA83 for 1 h at 4°C, and shifted for different times at 37°C. After immunoprecipitation with anti-HA beads, samples were analyzed by Western blotting using anti-Ub and anti-HA antibodies. (C) HeLa cells were transfected or untransfected with siRNAs against Cbl for 72 h and incubated with 500 ng/ml PA83 for different times at 37°C. Cell extracts were blotted for Cbl, tubulin (as an equal loading marker), and PA. (D) HeLa cells were untransfected or transfected with siRNAs against Cbl for a total of 72 h in total. 24 h later, these cells were additionally transfected with TEM8/1-HA for 48 h and incubated with 500 ng/ml PA83 for different times at 37°C. TEM8/1-HA was immunoprecipitated from each fraction and analyzed by SDS-PAGE and Western blotting using anti-Ub and anti-HA antibodies.

    Techniques Used: Transfection, Incubation, Immunoprecipitation, SDS Page, Western Blot, Marker

    5) Product Images from "Synthesis of Nanogels via Cell Membrane-Templated Polymerization"

    Article Title: Synthesis of Nanogels via Cell Membrane-Templated Polymerization

    Journal: Small (Weinheim an der Bergstrasse, Germany)

    doi: 10.1002/smll.201500987

    Confirming the formation of RBC membrane-coated nanogels. (A) Representative TEM images RBC membrane-coated nanogels and the nanogel cores after the treatment with Triton X-100 and proteinase K (scale bar = 100 nm). (B) RBC membrane-derived vesicles and
    Figure Legend Snippet: Confirming the formation of RBC membrane-coated nanogels. (A) Representative TEM images RBC membrane-coated nanogels and the nanogel cores after the treatment with Triton X-100 and proteinase K (scale bar = 100 nm). (B) RBC membrane-derived vesicles and

    Techniques Used: Transmission Electron Microscopy, Derivative Assay

    6) Product Images from "Enterohemorrhagic Escherichia coli O157:H7 Produces Tir, Which Is Translocated to the Host Cell Membrane but Is Not Tyrosine Phosphorylated"

    Article Title: Enterohemorrhagic Escherichia coli O157:H7 Produces Tir, Which Is Translocated to the Host Cell Membrane but Is Not Tyrosine Phosphorylated

    Journal: Infection and Immunity

    doi:

    EHEC Tir is translocated to the host cell but not tyrosine phosphorylated. HeLa cells were infected with either EHEC UMD619 or EPEC CVD206, and Triton X-100-soluble membrane fractions were prepared, treated with alkaline phosphatase (Alk Phos), and resolved by SDS–8% PAGE. After being blotted to nitrocellulose, the samples were probed with anti-EHEC Tir (a), anti-EPEC Tir (b), or anti-PY (c) antisera. Molecular mass markers are in kilodaltons.
    Figure Legend Snippet: EHEC Tir is translocated to the host cell but not tyrosine phosphorylated. HeLa cells were infected with either EHEC UMD619 or EPEC CVD206, and Triton X-100-soluble membrane fractions were prepared, treated with alkaline phosphatase (Alk Phos), and resolved by SDS–8% PAGE. After being blotted to nitrocellulose, the samples were probed with anti-EHEC Tir (a), anti-EPEC Tir (b), or anti-PY (c) antisera. Molecular mass markers are in kilodaltons.

    Techniques Used: Infection, Polyacrylamide Gel Electrophoresis

    7) Product Images from "Musashi-1 promotes a cancer stem cell lineage and chemoresistance in colorectal cancer cells"

    Article Title: Musashi-1 promotes a cancer stem cell lineage and chemoresistance in colorectal cancer cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-02057-9

    Musashi-1 forms SGs following treatment with arsenite. FLAGMusashi-1 transfected HT-29 cells were plated on coverslips and treated with 150 μM arsenite for 30 min. Cells were then fixed in 4% paraformaldehyde for 15 min at room temperature. After permeabilisation with 0.1% Triton X-100/PBS, FLAGMusashi-1-, PABP-, G3BP-, and eIF4E-specific antibodies were added to hybridization buffer at 4 °C overnight. The signals were amplified by Alexa488- or Alexa555-conjugated secondary antibodies. Images were acquired with a multiphoton confocal microscope. ( A ) Left panel: Co-localization of Musashi-1 and G3BP. Right panel: Statistical results of percentages of SG formation. Error bars indicate the mean ± SD from three independent experiments. # p
    Figure Legend Snippet: Musashi-1 forms SGs following treatment with arsenite. FLAGMusashi-1 transfected HT-29 cells were plated on coverslips and treated with 150 μM arsenite for 30 min. Cells were then fixed in 4% paraformaldehyde for 15 min at room temperature. After permeabilisation with 0.1% Triton X-100/PBS, FLAGMusashi-1-, PABP-, G3BP-, and eIF4E-specific antibodies were added to hybridization buffer at 4 °C overnight. The signals were amplified by Alexa488- or Alexa555-conjugated secondary antibodies. Images were acquired with a multiphoton confocal microscope. ( A ) Left panel: Co-localization of Musashi-1 and G3BP. Right panel: Statistical results of percentages of SG formation. Error bars indicate the mean ± SD from three independent experiments. # p

    Techniques Used: Transfection, Hybridization, Amplification, Microscopy

    8) Product Images from "Agonist Occupancy Is Essential for Forward Trafficking of AMPA Receptors"

    Article Title: Agonist Occupancy Is Essential for Forward Trafficking of AMPA Receptors

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3953-08.2009

    Secretion of GluR-D S1S2 ligand-binding domain constructs from transfected HEK293 cells.  A , Anti-Myc immunoblots of HEK293 growth media ( A ) and Triton X-100 extracts ( B ) of cells collected 40 h after transfection.  B , Quantification of the immunoblots. Ratios of secreted and cell-associated immunoreactivities are shown in relation to wt GluR-D i  S1S2, given an arbitrary value of 100%. The values and error bars correspond to a minimum of three independent experiments.
    Figure Legend Snippet: Secretion of GluR-D S1S2 ligand-binding domain constructs from transfected HEK293 cells. A , Anti-Myc immunoblots of HEK293 growth media ( A ) and Triton X-100 extracts ( B ) of cells collected 40 h after transfection. B , Quantification of the immunoblots. Ratios of secreted and cell-associated immunoreactivities are shown in relation to wt GluR-D i S1S2, given an arbitrary value of 100%. The values and error bars correspond to a minimum of three independent experiments.

    Techniques Used: Ligand Binding Assay, Construct, Transfection, Western Blot

    9) Product Images from "A Transmembrane Form of the Prion Protein Contains an Uncleaved Signal Peptide and Is Retained in the Endoplasmic Reticululm"

    Article Title: A Transmembrane Form of the Prion Protein Contains an Uncleaved Signal Peptide and Is Retained in the Endoplasmic Reticululm

    Journal: Molecular Biology of the Cell

    doi:

    Mutations in the transmembrane region  increase the proportion of  Ctm PrP, and reveal that this  form is slightly larger than  Sec PrP. mRNA encoding  wild-type (WT), A116V, or 3AV PrP was translated in rabbit reticulocyte  lysate supplemented with canine pancreatic microsomes. Aliquots of the  reaction were then incubated with (lanes 2, 3, 5, 6, 8, and 9) or  without (lanes 1, 4, and 7) PK in the presence (lanes 3, 6, and 9) or  absence (lanes 1, 2, 4, 5, 7, and 8) of Triton X-100 (Det). Samples  were then analyzed by SDS-PAGE and autoradiography. Note the presence  of a closely spaced doublet of glycosylated PrP in lanes 1, 4, and 7,  corresponding to  Sec PrP (white arrowheads) and  Ctm PrP (shaded arrowheads). The protease-protected  forms of  Sec PrP and  Ctm PrP are indicated by the  white and shaded arrows, respectively, in lanes 2, 5, and 8. Molecular  size markers are given in kilodaltons.
    Figure Legend Snippet: Mutations in the transmembrane region increase the proportion of Ctm PrP, and reveal that this form is slightly larger than Sec PrP. mRNA encoding wild-type (WT), A116V, or 3AV PrP was translated in rabbit reticulocyte lysate supplemented with canine pancreatic microsomes. Aliquots of the reaction were then incubated with (lanes 2, 3, 5, 6, 8, and 9) or without (lanes 1, 4, and 7) PK in the presence (lanes 3, 6, and 9) or absence (lanes 1, 2, 4, 5, 7, and 8) of Triton X-100 (Det). Samples were then analyzed by SDS-PAGE and autoradiography. Note the presence of a closely spaced doublet of glycosylated PrP in lanes 1, 4, and 7, corresponding to Sec PrP (white arrowheads) and Ctm PrP (shaded arrowheads). The protease-protected forms of Sec PrP and Ctm PrP are indicated by the white and shaded arrows, respectively, in lanes 2, 5, and 8. Molecular size markers are given in kilodaltons.

    Techniques Used: Size-exclusion Chromatography, Incubation, SDS Page, Autoradiography

    10) Product Images from "Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein"

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein

    Journal: Journal of Virology

    doi:

    Ability of mutant Rep proteins to introduce ITR plasmid into AAVS1. Two micrograms of pCMVR78, mutant Rep expression plasmids, or blank vector was transfected into 2 × 10 5  293 cells/well in six-well plates along with 2 μg of pW1, harboring a  lacZ  expression cassette flanked by ITRs, by a standard calcium phosphate precipitation method. Twenty-four hours later, total cellular DNA was isolated and suspended finally in 200 μl of TE. PCR to detect site-specific integration was carried out as reported previously with minor modifications: 1 μl of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH 4 ) 2 SO 4 , 4 mM MgSO 4 , 0.1% Triton X-100 (NEB)], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB). The cycling conditions were 99°C for 1 min followed by 35 cycles of 99°C for 10 s and 72°C for 4 min. Ten microliters of the PCR mixture was transferred to a hybridization membrane (Hybond-N + ; Amersham) by using a dot blot apparatus and hybridized with a  32 P-labeled AAVS1 probe. The membranes were then analyzed on a BAS-1500 imaging analyzer. The assay was repeated at least four times. p, positive control for hybridization.
    Figure Legend Snippet: Ability of mutant Rep proteins to introduce ITR plasmid into AAVS1. Two micrograms of pCMVR78, mutant Rep expression plasmids, or blank vector was transfected into 2 × 10 5 293 cells/well in six-well plates along with 2 μg of pW1, harboring a lacZ expression cassette flanked by ITRs, by a standard calcium phosphate precipitation method. Twenty-four hours later, total cellular DNA was isolated and suspended finally in 200 μl of TE. PCR to detect site-specific integration was carried out as reported previously with minor modifications: 1 μl of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH 4 ) 2 SO 4 , 4 mM MgSO 4 , 0.1% Triton X-100 (NEB)], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB). The cycling conditions were 99°C for 1 min followed by 35 cycles of 99°C for 10 s and 72°C for 4 min. Ten microliters of the PCR mixture was transferred to a hybridization membrane (Hybond-N + ; Amersham) by using a dot blot apparatus and hybridized with a 32 P-labeled AAVS1 probe. The membranes were then analyzed on a BAS-1500 imaging analyzer. The assay was repeated at least four times. p, positive control for hybridization.

    Techniques Used: Mutagenesis, Introduce, Plasmid Preparation, Expressing, Transfection, Isolation, Polymerase Chain Reaction, Hybridization, Dot Blot, Labeling, Imaging, Positive Control

    Related Articles

    Amplification:

    Article Title: Strong-LAMP: A LAMP Assay for Strongyloides spp. Detection in Stool and Urine Samples. Towards the Diagnosis of Human Strongyloidiasis Starting from a Rodent Model
    Article Snippet: .. Thus, LAMP reactions mixtures (25 μL) contained 40 pmol of each FIP and BIP primers, 5 pmol of each F3 and B3 primers, 1.4 mM of each dNTP (Bioron), 1x ThermoPol Reaction Buffer -20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100; New England Biolabs, UK- (when using Bst Polymerase Large Fragment) or 1x Isothermal Amplification Buffer -20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Tween20; New England Biolabs, UK- (when using either Bst DNA polymerase 2.0 or Bst DNA polymerase 2.0 WarmStart), betaine (ranging 0.8, 1, 1.2, 1.4 or 1.6 M), supplementary MgSO4 (ranging 2, 4, 6 or 8 mM) and 8 U of the tested Bst polymerase in each case with 2 μL of template DNA. .. All LAMP reactions mixtures were performed in 0.5-mL micro centrifuge tubes that were incubated in a heating block (K Dry-Bath) at a range of temperatures (61, 63 and 65°C) for 60 min to optimize the reaction conditions and then heated at 80°C for 5–10 min to inactivate the enzyme and thus to terminate the reaction.

    Article Title: Evaluating the influence of life‐history characteristics on genetic structure: a comparison of small mammals inhabiting complex agricultural landscapes
    Article Snippet: .. Amplification by multiplex PCR took place in 10 μ L volumes with 20 ng of template DNA, 0.2 mM of each dNTP, 1 U of Taq DNA polymerase (NEB), and 2× Thermopol reaction buffer (20 mM Tris‐HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X‐100; NEB). .. The amount of primer for each locus (0.05–0.30 μ M) was adjusted so that all loci in the multiplex reaction would result in approximately equal intensities of product.

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition
    Article Snippet: Paragraph title: PCR amplification of ssDNA library ... 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8, New England Biolabs).

    Article Title: Improved nucleotide selectivity and termination of 3?-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates
    Article Snippet: .. Approximately 50 ng of genomic DNA was amplified with 0.4 µM of HNF1a_2.1 (F/R) or HNF1a_10.1 (F/R) primer pairs and one unit of Vent(exo− ) polymerase in 1× ThermoPol buffer [20 mM Tris–HCl, pH 8.8; 10 mM (NH4 )2 SO4 ; 10 mM KCl; 2 mM MgSO4 ; 0.1% Triton X-100; New England BioLabs], 1 M betaine ( , ) and 200 μM each of dATP, dCTP, dGTP and either TTP or HOMedUTP. .. Cycling conditions were 4 min at 95°C, then 35 cycles of 95°C for 30 s, 59°C for 30 s and 72°C for 90 s with a final step at 72°C for seven min. PCR products were purified using a Qiagen QIAquick gel extraction kit and sequenced using a ‘1 /16 ’ dilution of BigDye version 3.1 chemistry.

    Article Title: Discerning non-disjunction in Down syndrome patients by means of GluK1-(AGAT)n and D21S2055-(GATA)n microsatellites on chromosome 21
    Article Snippet: [ ] PCR-based amplification of genomic DNA targets was carried out in the DNA Engine Thermal Cycler (MJ Research PTC-200). .. For the GluK1-(AGAT)n polymorphism, 5 pmol of each forward (5’-GCTAAATAGATATATGATAAACGG-3’) and reverse (5’-CTGGCAGTAAATGTCTATGAAAC-3’) primers[ ] were used in reactions containing 100 ng of template DNA, 1-X Thermopol-II buffer (NEB) containing of 10 mMKCl, 10 mM (NH4 )2 SO4, 20 mMTris-HCl (pH 8.8 at 25°C), 0.1% Triton X-100 (NEB), 1 mM MgSO4 , 200 μM dNTPs, and 0.2 U Taq DNA polymerase in 20μl reaction volume.

    Article Title: Mutational Disruption of cis-Acting Replication Element 2C in Coxsackievirus B3 Leads to 5′-Terminal Genomic Deletions
    Article Snippet: The two amplimers were gel purified, combined in Thermopol reaction buffer [20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 4 mM MgSO4 , 0.1% Triton X-100, pH 8.8; New England BioLabs] with 200 μM dNTPs, and repeatedly denatured and annealed 10 times (1 min at 94°C, 50°C for 1 min), followed by extension with Deep Vent polymerase (2,000 U/ml) at 72°C for 2 min. .. The mutated overlap extension product was subsequently directly amplified using the ID13 and XBA primers ( and ) and Deep Vent polymerase with the modified denaturation and annealing times described above.

    Article Title: Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane
    Article Snippet: Additionally, there were few or no amplification band(s) displayed after electrophoretic analysis ( ). .. The optimized LAMP reaction conditions including 4 mmol/L MgSO4 (50 mmol/L), 1.4 mmol/L dNTPs (10 mmol/L), 2.5 μL 10×ThermoPol Reaction Buffer [containing 20 mmol/L Tris-HCl pH 8.8, 10 mmol/L KCl, 2 mmol/L MgSO4 , 10 mmol/L (NH4 )2 SO4 , 0.1% Triton X-100, NEB company], 1.2 μmol/L each of FIP and BIP primers, 0.2 μmol/L each of F3 and B3 primers, 8.0 U Bst DNA polymerase (8 000 U/mL, NEB company), DNA template 1.0 μL, ddH2 O were complemented to a volume of 25 μL.

    Positive Control:

    Article Title: Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs
    Article Snippet: PCRs on undamaged DNA For the undamaged plasmid DNA template, 50 µl PCRs were set up containing standard ThermoPol reaction buffer [20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100] (New England Biolabs), 10 ng of circular pJM548 (Ste dpo4 -like in pGEM-T), 200 µM of each ultrapure dNTP and 1 µM of each primer; Ste_FNde5 and Ste_RBam1089 whose sequences are provided above. .. Aliquots containing 2.5 U of Taq DNA polymerase (New England Biolabs) were used as a positive control.

    Blocking Assay:

    Article Title: Strong-LAMP: A LAMP Assay for Strongyloides spp. Detection in Stool and Urine Samples. Towards the Diagnosis of Human Strongyloidiasis Starting from a Rodent Model
    Article Snippet: Thus, LAMP reactions mixtures (25 μL) contained 40 pmol of each FIP and BIP primers, 5 pmol of each F3 and B3 primers, 1.4 mM of each dNTP (Bioron), 1x ThermoPol Reaction Buffer -20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100; New England Biolabs, UK- (when using Bst Polymerase Large Fragment) or 1x Isothermal Amplification Buffer -20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Tween20; New England Biolabs, UK- (when using either Bst DNA polymerase 2.0 or Bst DNA polymerase 2.0 WarmStart), betaine (ranging 0.8, 1, 1.2, 1.4 or 1.6 M), supplementary MgSO4 (ranging 2, 4, 6 or 8 mM) and 8 U of the tested Bst polymerase in each case with 2 μL of template DNA. .. All LAMP reactions mixtures were performed in 0.5-mL micro centrifuge tubes that were incubated in a heating block (K Dry-Bath) at a range of temperatures (61, 63 and 65°C) for 60 min to optimize the reaction conditions and then heated at 80°C for 5–10 min to inactivate the enzyme and thus to terminate the reaction.

    SYBR Green Assay:

    Article Title: Development of a loop-mediated isothermal amplification method to rapidly detect porcine circovirus genotypes 2a and 2b
    Article Snippet: Bst DNA polymerase and 10 × ThermoPolbuffer (20 mM Tris–HCl, 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100) were purchased from New England Biolabs (Massachusetts, USA). .. SYBR Green I dye was obtained from Solar Biotechnology (Beijing, China).

    Article Title: Rapid and sensitive detection of Pseudocercospora eumusae pathogen causing eumusae leaf spot disease of banana by loop-mediated isothermal amplification (LAMP) method
    Article Snippet: To determine the optimal reaction temperature and time, the LAMP reaction mixtures were incubated at 56–65 °C for 15, 30, 45, 60, and 90 min. A total volume of 25 ul for the LAMP reaction contained 0.48 µM FIP and BIP, 0.12 µM concentrations of F3 and B3, 2 mM dNTPs mix, 2.0 U of Bst DNA polymerase large fragment (New England Biolabs, Inc., Beverly, MA), 1× ThermoPol buffer (20 mM Tris–HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8) (New England Biolabs), 4 mM MgSO4 and 1 µl of template DNA (25 ng). .. The visual detection of LAMP products was performed by the addition of 2 µl of 1000× SYBR® Green I nucleic acid gel stain (Invitrogen, Carlsbad, California).

    Article Title: Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane
    Article Snippet: As shown in , combinations 1, 2, 5, 6, 11 and 16 exhibited no or extremely weak flavogreen fluorescent signals in the SYBR Green I test. .. The optimized LAMP reaction conditions including 4 mmol/L MgSO4 (50 mmol/L), 1.4 mmol/L dNTPs (10 mmol/L), 2.5 μL 10×ThermoPol Reaction Buffer [containing 20 mmol/L Tris-HCl pH 8.8, 10 mmol/L KCl, 2 mmol/L MgSO4 , 10 mmol/L (NH4 )2 SO4 , 0.1% Triton X-100, NEB company], 1.2 μmol/L each of FIP and BIP primers, 0.2 μmol/L each of F3 and B3 primers, 8.0 U Bst DNA polymerase (8 000 U/mL, NEB company), DNA template 1.0 μL, ddH2 O were complemented to a volume of 25 μL.

    Incubation:

    Article Title: Strong-LAMP: A LAMP Assay for Strongyloides spp. Detection in Stool and Urine Samples. Towards the Diagnosis of Human Strongyloidiasis Starting from a Rodent Model
    Article Snippet: Thus, LAMP reactions mixtures (25 μL) contained 40 pmol of each FIP and BIP primers, 5 pmol of each F3 and B3 primers, 1.4 mM of each dNTP (Bioron), 1x ThermoPol Reaction Buffer -20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100; New England Biolabs, UK- (when using Bst Polymerase Large Fragment) or 1x Isothermal Amplification Buffer -20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Tween20; New England Biolabs, UK- (when using either Bst DNA polymerase 2.0 or Bst DNA polymerase 2.0 WarmStart), betaine (ranging 0.8, 1, 1.2, 1.4 or 1.6 M), supplementary MgSO4 (ranging 2, 4, 6 or 8 mM) and 8 U of the tested Bst polymerase in each case with 2 μL of template DNA. .. All LAMP reactions mixtures were performed in 0.5-mL micro centrifuge tubes that were incubated in a heating block (K Dry-Bath) at a range of temperatures (61, 63 and 65°C) for 60 min to optimize the reaction conditions and then heated at 80°C for 5–10 min to inactivate the enzyme and thus to terminate the reaction.

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: The samples were mixed with 20 μg of proteinase K, incubated for 6 h at 55°C, extracted with an equal volume of phenol-chloroform, ethanol precipitated, and then suspended in 200 μl of TE. .. One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB).

    Article Title: Rapid and sensitive detection of Pseudocercospora eumusae pathogen causing eumusae leaf spot disease of banana by loop-mediated isothermal amplification (LAMP) method
    Article Snippet: .. To determine the optimal reaction temperature and time, the LAMP reaction mixtures were incubated at 56–65 °C for 15, 30, 45, 60, and 90 min. A total volume of 25 ul for the LAMP reaction contained 0.48 µM FIP and BIP, 0.12 µM concentrations of F3 and B3, 2 mM dNTPs mix, 2.0 U of Bst DNA polymerase large fragment (New England Biolabs, Inc., Beverly, MA), 1× ThermoPol buffer (20 mM Tris–HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8) (New England Biolabs), 4 mM MgSO4 and 1 µl of template DNA (25 ng). .. The visual detection of LAMP products was performed by the addition of 2 µl of 1000× SYBR® Green I nucleic acid gel stain (Invitrogen, Carlsbad, California).

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: The primer–template complex was annealed in ThermoPol buffer (1 × : 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8; New England Biolabs) by heating for 5 min at 95 °C and cooling for 5 min at 4 °C. .. Nucleotide triphosphates (100 μM final) and polymerase were added to the reaction after primer annealing and the reaction was incubated at 55 °C for the indicated amount of time.

    Gel Extraction:

    Article Title: Improved nucleotide selectivity and termination of 3?-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates
    Article Snippet: Approximately 50 ng of genomic DNA was amplified with 0.4 µM of HNF1a_2.1 (F/R) or HNF1a_10.1 (F/R) primer pairs and one unit of Vent(exo− ) polymerase in 1× ThermoPol buffer [20 mM Tris–HCl, pH 8.8; 10 mM (NH4 )2 SO4 ; 10 mM KCl; 2 mM MgSO4 ; 0.1% Triton X-100; New England BioLabs], 1 M betaine ( , ) and 200 μM each of dATP, dCTP, dGTP and either TTP or HOMedUTP. .. Cycling conditions were 4 min at 95°C, then 35 cycles of 95°C for 30 s, 59°C for 30 s and 72°C for 90 s with a final step at 72°C for seven min. PCR products were purified using a Qiagen QIAquick gel extraction kit and sequenced using a ‘1 /16 ’ dilution of BigDye version 3.1 chemistry.

    Activity Assay:

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: Paragraph title: Polymerase activity assays ... The primer–template complex was annealed in ThermoPol buffer (1 × : 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8; New England Biolabs) by heating for 5 min at 95 °C and cooling for 5 min at 4 °C.

    Expressing:

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: Two micrograms of pCMVR78, mutant Rep expression plasmid, or blank vector was transfected into 2 × 105 293 cells/well in six-well plates along with 2 μg of pW1, harboring a lacZ expression cassette flanked by ITRs, by a standard calcium phosphate precipitation method. .. One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB).

    Modification:

    Article Title: Evaluating the influence of life‐history characteristics on genetic structure: a comparison of small mammals inhabiting complex agricultural landscapes
    Article Snippet: Microsatellite genotyping We used an ammonium acetate protocol and an ethanol wash to extract DNA from tissue samples (modified from the PUREGENE kit; Gentra Systems, Minneapolis, MN). .. Amplification by multiplex PCR took place in 10 μ L volumes with 20 ng of template DNA, 0.2 mM of each dNTP, 1 U of Taq DNA polymerase (NEB), and 2× Thermopol reaction buffer (20 mM Tris‐HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X‐100; NEB).

    Article Title: Mutational Disruption of cis-Acting Replication Element 2C in Coxsackievirus B3 Leads to 5′-Terminal Genomic Deletions
    Article Snippet: Cycling was carried out as noted in with modified cycle denaturation and annealing times of 30 s each. .. The two amplimers were gel purified, combined in Thermopol reaction buffer [20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 4 mM MgSO4 , 0.1% Triton X-100, pH 8.8; New England BioLabs] with 200 μM dNTPs, and repeatedly denatured and annealed 10 times (1 min at 94°C, 50°C for 1 min), followed by extension with Deep Vent polymerase (2,000 U/ml) at 72°C for 2 min.

    Derivative Assay:

    Article Title: Rapid and sensitive detection of Pseudocercospora eumusae pathogen causing eumusae leaf spot disease of banana by loop-mediated isothermal amplification (LAMP) method
    Article Snippet: The LAMP primers specific to P. eumusae were designed based on the specific SCAR marker sequence of P. eumusae derived from RAPD analysis. .. To determine the optimal reaction temperature and time, the LAMP reaction mixtures were incubated at 56–65 °C for 15, 30, 45, 60, and 90 min. A total volume of 25 ul for the LAMP reaction contained 0.48 µM FIP and BIP, 0.12 µM concentrations of F3 and B3, 2 mM dNTPs mix, 2.0 U of Bst DNA polymerase large fragment (New England Biolabs, Inc., Beverly, MA), 1× ThermoPol buffer (20 mM Tris–HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8) (New England Biolabs), 4 mM MgSO4 and 1 µl of template DNA (25 ng).

    Hybridization:

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB). .. Ten microliters of the PCR mixture was transferred to a hybridization membrane (Hybond-N+; Amersham) by using a dot blot apparatus and hybridized with an AAVS1 probe.

    High Performance Liquid Chromatography:

    Article Title: Improved nucleotide selectivity and termination of 3?-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates
    Article Snippet: Approximately 50 ng of genomic DNA was amplified with 0.4 µM of HNF1a_2.1 (F/R) or HNF1a_10.1 (F/R) primer pairs and one unit of Vent(exo− ) polymerase in 1× ThermoPol buffer [20 mM Tris–HCl, pH 8.8; 10 mM (NH4 )2 SO4 ; 10 mM KCl; 2 mM MgSO4 ; 0.1% Triton X-100; New England BioLabs], 1 M betaine ( , ) and 200 μM each of dATP, dCTP, dGTP and either TTP or HOMedUTP. .. Reaction products were purified by ethanol precipitation, resuspended in 30 µl of HPLC water, and then loaded onto an AB model 3100 DNA sequencer.

    Transfection:

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: Two micrograms of pCMVR78, mutant Rep expression plasmid, or blank vector was transfected into 2 × 105 293 cells/well in six-well plates along with 2 μg of pW1, harboring a lacZ expression cassette flanked by ITRs, by a standard calcium phosphate precipitation method. .. One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB).

    Generated:

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB). .. The probe was a random-primed 32 P-labeled PCR fragment generated in a reaction with 20 ng of pRVK as a template and primers 5′-ACTTTGAGCTCTACTGGCTTC and 5′-GGAGGATCCGCTCAGAGG.

    Article Title: Mutational Disruption of cis-Acting Replication Element 2C in Coxsackievirus B3 Leads to 5′-Terminal Genomic Deletions
    Article Snippet: Two overlapping fragments of CVB3 cDNA were generated from the infectious cDNA clone of CVB3/28 ( ) by PCR using Deep Vent DNA polymerase (New England BioLabs, Ipswich, MA) and the primer pair ID13 and CREKORev or CREKO and XBA ( ) with 4 mM MgSO4 and 200 μM deoxynucleoside triphosphates (dNTPs). .. The two amplimers were gel purified, combined in Thermopol reaction buffer [20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 4 mM MgSO4 , 0.1% Triton X-100, pH 8.8; New England BioLabs] with 200 μM dNTPs, and repeatedly denatured and annealed 10 times (1 min at 94°C, 50°C for 1 min), followed by extension with Deep Vent polymerase (2,000 U/ml) at 72°C for 2 min.

    Imaging:

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB). .. The membranes were then analyzed on a BAS-1500 imaging analyzer.

    Isolation:

    Article Title: Discerning non-disjunction in Down syndrome patients by means of GluK1-(AGAT)n and D21S2055-(GATA)n microsatellites on chromosome 21
    Article Snippet: Genotyping procedures Genomic DNA was isolated from whole blood lymphocytes by the salting-out procedure. .. For the GluK1-(AGAT)n polymorphism, 5 pmol of each forward (5’-GCTAAATAGATATATGATAAACGG-3’) and reverse (5’-CTGGCAGTAAATGTCTATGAAAC-3’) primers[ ] were used in reactions containing 100 ng of template DNA, 1-X Thermopol-II buffer (NEB) containing of 10 mMKCl, 10 mM (NH4 )2 SO4, 20 mMTris-HCl (pH 8.8 at 25°C), 0.1% Triton X-100 (NEB), 1 mM MgSO4 , 200 μM dNTPs, and 0.2 U Taq DNA polymerase in 20μl reaction volume.

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: .. One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB). .. Ten microliters of the PCR mixture was transferred to a hybridization membrane (Hybond-N+; Amersham) by using a dot blot apparatus and hybridized with an AAVS1 probe.

    Sequencing:

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition
    Article Snippet: A single stranded DNA library having the sequence 5′-GCGCATACCAGCTTATTCAATT-N50 -AGATAGTAAGTGCAATCTCGGC-3′ (20 pmol) was amplified in 50 μl PCR reactions containing 0.2 μM template, 0.2 μM each primers, 200 μM dNTPs, and 2.5 U Taq DNA polymerase in 1× Thermopol buffer (20 mM Tris–HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl. .. 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8, New England Biolabs).

    Article Title: Improved nucleotide selectivity and termination of 3?-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates
    Article Snippet: Paragraph title: PCR and Sanger sequencing ... Approximately 50 ng of genomic DNA was amplified with 0.4 µM of HNF1a_2.1 (F/R) or HNF1a_10.1 (F/R) primer pairs and one unit of Vent(exo− ) polymerase in 1× ThermoPol buffer [20 mM Tris–HCl, pH 8.8; 10 mM (NH4 )2 SO4 ; 10 mM KCl; 2 mM MgSO4 ; 0.1% Triton X-100; New England BioLabs], 1 M betaine ( , ) and 200 μM each of dATP, dCTP, dGTP and either TTP or HOMedUTP.

    Article Title: Rapid and sensitive detection of Pseudocercospora eumusae pathogen causing eumusae leaf spot disease of banana by loop-mediated isothermal amplification (LAMP) method
    Article Snippet: The LAMP primers specific to P. eumusae were designed based on the specific SCAR marker sequence of P. eumusae derived from RAPD analysis. .. To determine the optimal reaction temperature and time, the LAMP reaction mixtures were incubated at 56–65 °C for 15, 30, 45, 60, and 90 min. A total volume of 25 ul for the LAMP reaction contained 0.48 µM FIP and BIP, 0.12 µM concentrations of F3 and B3, 2 mM dNTPs mix, 2.0 U of Bst DNA polymerase large fragment (New England Biolabs, Inc., Beverly, MA), 1× ThermoPol buffer (20 mM Tris–HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8) (New England Biolabs), 4 mM MgSO4 and 1 µl of template DNA (25 ng).

    Fluorescence:

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: Primer-extension reactions were analysed by denaturing PAGE or fluorescence. .. The primer–template complex was annealed in ThermoPol buffer (1 × : 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8; New England Biolabs) by heating for 5 min at 95 °C and cooling for 5 min at 4 °C.

    Article Title: Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane
    Article Snippet: LAMP products of combination 7 showed high flavogreen fluorescence, and displayed bright typical ladder-like bands after electrophoresis, apparent in distinction from the negative and blank controls, whereas combinations 4, 8 and 10 showed intensive flavogreen fluorescent signals but weaker band brightness than combination 7 in electrophoresis. .. The optimized LAMP reaction conditions including 4 mmol/L MgSO4 (50 mmol/L), 1.4 mmol/L dNTPs (10 mmol/L), 2.5 μL 10×ThermoPol Reaction Buffer [containing 20 mmol/L Tris-HCl pH 8.8, 10 mmol/L KCl, 2 mmol/L MgSO4 , 10 mmol/L (NH4 )2 SO4 , 0.1% Triton X-100, NEB company], 1.2 μmol/L each of FIP and BIP primers, 0.2 μmol/L each of F3 and B3 primers, 8.0 U Bst DNA polymerase (8 000 U/mL, NEB company), DNA template 1.0 μL, ddH2 O were complemented to a volume of 25 μL.

    Mutagenesis:

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: Two micrograms of pCMVR78, mutant Rep expression plasmid, or blank vector was transfected into 2 × 105 293 cells/well in six-well plates along with 2 μg of pW1, harboring a lacZ expression cassette flanked by ITRs, by a standard calcium phosphate precipitation method. .. One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB).

    Article Title: Mutational Disruption of cis-Acting Replication Element 2C in Coxsackievirus B3 Leads to 5′-Terminal Genomic Deletions
    Article Snippet: The mutations in the CVB3 CRE(2C) were generated using overlap extension mutagenesis ( ). .. The two amplimers were gel purified, combined in Thermopol reaction buffer [20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 4 mM MgSO4 , 0.1% Triton X-100, pH 8.8; New England BioLabs] with 200 μM dNTPs, and repeatedly denatured and annealed 10 times (1 min at 94°C, 50°C for 1 min), followed by extension with Deep Vent polymerase (2,000 U/ml) at 72°C for 2 min.

    Random Primed:

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB). .. The probe was a random-primed 32 P-labeled PCR fragment generated in a reaction with 20 ng of pRVK as a template and primers 5′-ACTTTGAGCTCTACTGGCTTC and 5′-GGAGGATCCGCTCAGAGG.

    Negative Control:

    Article Title: Strong-LAMP: A LAMP Assay for Strongyloides spp. Detection in Stool and Urine Samples. Towards the Diagnosis of Human Strongyloidiasis Starting from a Rodent Model
    Article Snippet: Thus, LAMP reactions mixtures (25 μL) contained 40 pmol of each FIP and BIP primers, 5 pmol of each F3 and B3 primers, 1.4 mM of each dNTP (Bioron), 1x ThermoPol Reaction Buffer -20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100; New England Biolabs, UK- (when using Bst Polymerase Large Fragment) or 1x Isothermal Amplification Buffer -20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Tween20; New England Biolabs, UK- (when using either Bst DNA polymerase 2.0 or Bst DNA polymerase 2.0 WarmStart), betaine (ranging 0.8, 1, 1.2, 1.4 or 1.6 M), supplementary MgSO4 (ranging 2, 4, 6 or 8 mM) and 8 U of the tested Bst polymerase in each case with 2 μL of template DNA. .. Template DNA was replaced by ultrapure water as negative control in each LAMP reaction.

    Labeling:

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition
    Article Snippet: 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8, New England Biolabs). .. The amplified double stranded DNA was purified using a PCR cleanup column (Qiagen) and the PEG-functionalized strand was separated from the FAM labeled strand on a denaturing 10% polyacrylamide gel.

    Mouse Assay:

    Article Title: Evaluating the influence of life‐history characteristics on genetic structure: a comparison of small mammals inhabiting complex agricultural landscapes
    Article Snippet: DNA quality was checked by running 3 μ L of DNA out on a 2% agarose gel stained with ethidium 12 bromide, and then, DNA extracts were diluted to a final concentration of approximately 10 ng/μ L. Chipmunk samples were amplified at 12 (EACH01‐12; Anderson et al. ) microsatellite loci, while white‐footed mice were amplified at 10 loci [PO‐26, PO‐85, Po‐97 (Prince et al. ); Pml01, Pml02, Pml05, Pml09, Pml12 (Chirhart et al. ); PLGT15 (Schmidt )]. .. Amplification by multiplex PCR took place in 10 μ L volumes with 20 ng of template DNA, 0.2 mM of each dNTP, 1 U of Taq DNA polymerase (NEB), and 2× Thermopol reaction buffer (20 mM Tris‐HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X‐100; NEB).

    Dot Blot:

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB). .. Ten microliters of the PCR mixture was transferred to a hybridization membrane (Hybond-N+; Amersham) by using a dot blot apparatus and hybridized with an AAVS1 probe.

    Polymerase Chain Reaction:

    Article Title: Evaluating the influence of life‐history characteristics on genetic structure: a comparison of small mammals inhabiting complex agricultural landscapes
    Article Snippet: .. Amplification by multiplex PCR took place in 10 μ L volumes with 20 ng of template DNA, 0.2 mM of each dNTP, 1 U of Taq DNA polymerase (NEB), and 2× Thermopol reaction buffer (20 mM Tris‐HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X‐100; NEB). .. The amount of primer for each locus (0.05–0.30 μ M) was adjusted so that all loci in the multiplex reaction would result in approximately equal intensities of product.

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition
    Article Snippet: Paragraph title: PCR amplification of ssDNA library ... 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8, New England Biolabs).

    Article Title: Improved nucleotide selectivity and termination of 3?-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates
    Article Snippet: Paragraph title: PCR and Sanger sequencing ... Approximately 50 ng of genomic DNA was amplified with 0.4 µM of HNF1a_2.1 (F/R) or HNF1a_10.1 (F/R) primer pairs and one unit of Vent(exo− ) polymerase in 1× ThermoPol buffer [20 mM Tris–HCl, pH 8.8; 10 mM (NH4 )2 SO4 ; 10 mM KCl; 2 mM MgSO4 ; 0.1% Triton X-100; New England BioLabs], 1 M betaine ( , ) and 200 μM each of dATP, dCTP, dGTP and either TTP or HOMedUTP.

    Article Title: Discerning non-disjunction in Down syndrome patients by means of GluK1-(AGAT)n and D21S2055-(GATA)n microsatellites on chromosome 21
    Article Snippet: [ ] PCR-based amplification of genomic DNA targets was carried out in the DNA Engine Thermal Cycler (MJ Research PTC-200). .. For the GluK1-(AGAT)n polymorphism, 5 pmol of each forward (5’-GCTAAATAGATATATGATAAACGG-3’) and reverse (5’-CTGGCAGTAAATGTCTATGAAAC-3’) primers[ ] were used in reactions containing 100 ng of template DNA, 1-X Thermopol-II buffer (NEB) containing of 10 mMKCl, 10 mM (NH4 )2 SO4, 20 mMTris-HCl (pH 8.8 at 25°C), 0.1% Triton X-100 (NEB), 1 mM MgSO4 , 200 μM dNTPs, and 0.2 U Taq DNA polymerase in 20μl reaction volume.

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: Paragraph title: PCR-based assay for site-specific integration at the AAVS1 locus. ... One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB).

    Article Title: Mutational Disruption of cis-Acting Replication Element 2C in Coxsackievirus B3 Leads to 5′-Terminal Genomic Deletions
    Article Snippet: Paragraph title: Mutational disruption of CRE(2C) using overlap extension PCR. ... The two amplimers were gel purified, combined in Thermopol reaction buffer [20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 4 mM MgSO4 , 0.1% Triton X-100, pH 8.8; New England BioLabs] with 200 μM dNTPs, and repeatedly denatured and annealed 10 times (1 min at 94°C, 50°C for 1 min), followed by extension with Deep Vent polymerase (2,000 U/ml) at 72°C for 2 min.

    Polyacrylamide Gel Electrophoresis:

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: Primer-extension reactions were analysed by denaturing PAGE or fluorescence. .. The primer–template complex was annealed in ThermoPol buffer (1 × : 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8; New England Biolabs) by heating for 5 min at 95 °C and cooling for 5 min at 4 °C.

    Salting Out:

    Article Title: Discerning non-disjunction in Down syndrome patients by means of GluK1-(AGAT)n and D21S2055-(GATA)n microsatellites on chromosome 21
    Article Snippet: Genotyping procedures Genomic DNA was isolated from whole blood lymphocytes by the salting-out procedure. .. For the GluK1-(AGAT)n polymorphism, 5 pmol of each forward (5’-GCTAAATAGATATATGATAAACGG-3’) and reverse (5’-CTGGCAGTAAATGTCTATGAAAC-3’) primers[ ] were used in reactions containing 100 ng of template DNA, 1-X Thermopol-II buffer (NEB) containing of 10 mMKCl, 10 mM (NH4 )2 SO4, 20 mMTris-HCl (pH 8.8 at 25°C), 0.1% Triton X-100 (NEB), 1 mM MgSO4 , 200 μM dNTPs, and 0.2 U Taq DNA polymerase in 20μl reaction volume.

    Purification:

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition
    Article Snippet: 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8, New England Biolabs). .. The amplified double stranded DNA was purified using a PCR cleanup column (Qiagen) and the PEG-functionalized strand was separated from the FAM labeled strand on a denaturing 10% polyacrylamide gel.

    Article Title: Improved nucleotide selectivity and termination of 3?-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates
    Article Snippet: Approximately 50 ng of genomic DNA was amplified with 0.4 µM of HNF1a_2.1 (F/R) or HNF1a_10.1 (F/R) primer pairs and one unit of Vent(exo− ) polymerase in 1× ThermoPol buffer [20 mM Tris–HCl, pH 8.8; 10 mM (NH4 )2 SO4 ; 10 mM KCl; 2 mM MgSO4 ; 0.1% Triton X-100; New England BioLabs], 1 M betaine ( , ) and 200 μM each of dATP, dCTP, dGTP and either TTP or HOMedUTP. .. Cycling conditions were 4 min at 95°C, then 35 cycles of 95°C for 30 s, 59°C for 30 s and 72°C for 90 s with a final step at 72°C for seven min. PCR products were purified using a Qiagen QIAquick gel extraction kit and sequenced using a ‘1 /16 ’ dilution of BigDye version 3.1 chemistry.

    Article Title: Mutational Disruption of cis-Acting Replication Element 2C in Coxsackievirus B3 Leads to 5′-Terminal Genomic Deletions
    Article Snippet: .. The two amplimers were gel purified, combined in Thermopol reaction buffer [20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 4 mM MgSO4 , 0.1% Triton X-100, pH 8.8; New England BioLabs] with 200 μM dNTPs, and repeatedly denatured and annealed 10 times (1 min at 94°C, 50°C for 1 min), followed by extension with Deep Vent polymerase (2,000 U/ml) at 72°C for 2 min. .. The mutated overlap extension product was subsequently directly amplified using the ID13 and XBA primers ( and ) and Deep Vent polymerase with the modified denaturation and annealing times described above.

    Plasmid Preparation:

    Article Title: Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs
    Article Snippet: .. PCRs on undamaged DNA For the undamaged plasmid DNA template, 50 µl PCRs were set up containing standard ThermoPol reaction buffer [20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100] (New England Biolabs), 10 ng of circular pJM548 (Ste dpo4 -like in pGEM-T), 200 µM of each ultrapure dNTP and 1 µM of each primer; Ste_FNde5 and Ste_RBam1089 whose sequences are provided above. .. Aliquots containing 2.5 U of Taq DNA polymerase (New England Biolabs) were used as a positive control.

    Article Title: Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
    Article Snippet: Two micrograms of pCMVR78, mutant Rep expression plasmid, or blank vector was transfected into 2 × 105 293 cells/well in six-well plates along with 2 μg of pW1, harboring a lacZ expression cassette flanked by ITRs, by a standard calcium phosphate precipitation method. .. One microliter of isolated genomic DNA was subjected to a thermal cycling reaction in a 20-μl reaction mixture containing 1× thermophilic DNA polymerase buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100 (New England Biolabs {NEB})], 1 μM 5′-CGGCCTCAGTGAGCGAGCGAGC and 5′-CGGGGAGGATCCGCTCAGAGGACA, and 2 U of Deep Vent Exo(−) DNA polymerase (NEB).

    Software:

    Article Title: Evaluating the influence of life‐history characteristics on genetic structure: a comparison of small mammals inhabiting complex agricultural landscapes
    Article Snippet: Amplification by multiplex PCR took place in 10 μ L volumes with 20 ng of template DNA, 0.2 mM of each dNTP, 1 U of Taq DNA polymerase (NEB), and 2× Thermopol reaction buffer (20 mM Tris‐HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X‐100; NEB). .. The PCR products were sized on an Applied Biosystems 3730 automated sequencer, and the genotypes were determined for all loci in all individuals using the software GeneMapper 3.7 (Applied Biosystems, Foster City, CA).

    Electrophoresis:

    Article Title: Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane
    Article Snippet: LAMP products of combination 7 showed high flavogreen fluorescence, and displayed bright typical ladder-like bands after electrophoresis, apparent in distinction from the negative and blank controls, whereas combinations 4, 8 and 10 showed intensive flavogreen fluorescent signals but weaker band brightness than combination 7 in electrophoresis. .. The optimized LAMP reaction conditions including 4 mmol/L MgSO4 (50 mmol/L), 1.4 mmol/L dNTPs (10 mmol/L), 2.5 μL 10×ThermoPol Reaction Buffer [containing 20 mmol/L Tris-HCl pH 8.8, 10 mmol/L KCl, 2 mmol/L MgSO4 , 10 mmol/L (NH4 )2 SO4 , 0.1% Triton X-100, NEB company], 1.2 μmol/L each of FIP and BIP primers, 0.2 μmol/L each of F3 and B3 primers, 8.0 U Bst DNA polymerase (8 000 U/mL, NEB company), DNA template 1.0 μL, ddH2 O were complemented to a volume of 25 μL.

    Multiplex Assay:

    Article Title: Evaluating the influence of life‐history characteristics on genetic structure: a comparison of small mammals inhabiting complex agricultural landscapes
    Article Snippet: .. Amplification by multiplex PCR took place in 10 μ L volumes with 20 ng of template DNA, 0.2 mM of each dNTP, 1 U of Taq DNA polymerase (NEB), and 2× Thermopol reaction buffer (20 mM Tris‐HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X‐100; NEB). .. The amount of primer for each locus (0.05–0.30 μ M) was adjusted so that all loci in the multiplex reaction would result in approximately equal intensities of product.

    Agarose Gel Electrophoresis:

    Article Title: Evaluating the influence of life‐history characteristics on genetic structure: a comparison of small mammals inhabiting complex agricultural landscapes
    Article Snippet: DNA quality was checked by running 3 μ L of DNA out on a 2% agarose gel stained with ethidium 12 bromide, and then, DNA extracts were diluted to a final concentration of approximately 10 ng/μ L. Chipmunk samples were amplified at 12 (EACH01‐12; Anderson et al. ) microsatellite loci, while white‐footed mice were amplified at 10 loci [PO‐26, PO‐85, Po‐97 (Prince et al. ); Pml01, Pml02, Pml05, Pml09, Pml12 (Chirhart et al. ); PLGT15 (Schmidt )]. .. Amplification by multiplex PCR took place in 10 μ L volumes with 20 ng of template DNA, 0.2 mM of each dNTP, 1 U of Taq DNA polymerase (NEB), and 2× Thermopol reaction buffer (20 mM Tris‐HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X‐100; NEB).

    Article Title: Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs
    Article Snippet: PCRs on undamaged DNA For the undamaged plasmid DNA template, 50 µl PCRs were set up containing standard ThermoPol reaction buffer [20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100] (New England Biolabs), 10 ng of circular pJM548 (Ste dpo4 -like in pGEM-T), 200 µM of each ultrapure dNTP and 1 µM of each primer; Ste_FNde5 and Ste_RBam1089 whose sequences are provided above. .. Reactions were heated to 85°C for 3 min and then treated with 35 cycles of 85°C for 30 s, 56°C for 1 min and 60°C for 4 min. Products were separated on a 0.9% agarose gel stained with ethidium bromide.

    Size-exclusion Chromatography:

    Article Title: Discerning non-disjunction in Down syndrome patients by means of GluK1-(AGAT)n and D21S2055-(GATA)n microsatellites on chromosome 21
    Article Snippet: For the GluK1-(AGAT)n polymorphism, 5 pmol of each forward (5’-GCTAAATAGATATATGATAAACGG-3’) and reverse (5’-CTGGCAGTAAATGTCTATGAAAC-3’) primers[ ] were used in reactions containing 100 ng of template DNA, 1-X Thermopol-II buffer (NEB) containing of 10 mMKCl, 10 mM (NH4 )2 SO4, 20 mMTris-HCl (pH 8.8 at 25°C), 0.1% Triton X-100 (NEB), 1 mM MgSO4 , 200 μM dNTPs, and 0.2 U Taq DNA polymerase in 20μl reaction volume. .. The cycling conditions were as follows: Denaturation at 95°C for 10 min followed by 30 cycles of denaturation at 95°C for 40 sec; annealing at 58°C for 40 sec; elongation at 72°C for 40 sec; final elongation step at 72°C for 7 min. Amplicons and size standards were resolved in 12% polyacrylamide gels containing 5% glycerol.

    Ethanol Precipitation:

    Article Title: Improved nucleotide selectivity and termination of 3?-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates
    Article Snippet: Approximately 50 ng of genomic DNA was amplified with 0.4 µM of HNF1a_2.1 (F/R) or HNF1a_10.1 (F/R) primer pairs and one unit of Vent(exo− ) polymerase in 1× ThermoPol buffer [20 mM Tris–HCl, pH 8.8; 10 mM (NH4 )2 SO4 ; 10 mM KCl; 2 mM MgSO4 ; 0.1% Triton X-100; New England BioLabs], 1 M betaine ( , ) and 200 μM each of dATP, dCTP, dGTP and either TTP or HOMedUTP. .. Reaction products were purified by ethanol precipitation, resuspended in 30 µl of HPLC water, and then loaded onto an AB model 3100 DNA sequencer.

    Concentration Assay:

    Article Title: Strong-LAMP: A LAMP Assay for Strongyloides spp. Detection in Stool and Urine Samples. Towards the Diagnosis of Human Strongyloidiasis Starting from a Rodent Model
    Article Snippet: Optimization of LAMP assay We tried to evaluate the LAMP primer set designed by using different in house reaction mixtures each containing a different Bst polymerase (namely, Bst DNA polymerase Large Fragment, Bst DNA polymerase 2.0 and Bst DNA polymerase 2.0 WarmStart; New England Biolabs, UK) as well as varying concentration of betaine (Sigma, USA) and supplementary MgSO4 (New England Biolabs, UK) to compare results in S . venezuelensis DNA amplification. .. Thus, LAMP reactions mixtures (25 μL) contained 40 pmol of each FIP and BIP primers, 5 pmol of each F3 and B3 primers, 1.4 mM of each dNTP (Bioron), 1x ThermoPol Reaction Buffer -20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100; New England Biolabs, UK- (when using Bst Polymerase Large Fragment) or 1x Isothermal Amplification Buffer -20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Tween20; New England Biolabs, UK- (when using either Bst DNA polymerase 2.0 or Bst DNA polymerase 2.0 WarmStart), betaine (ranging 0.8, 1, 1.2, 1.4 or 1.6 M), supplementary MgSO4 (ranging 2, 4, 6 or 8 mM) and 8 U of the tested Bst polymerase in each case with 2 μL of template DNA.

    Article Title: Evaluating the influence of life‐history characteristics on genetic structure: a comparison of small mammals inhabiting complex agricultural landscapes
    Article Snippet: DNA quality was checked by running 3 μ L of DNA out on a 2% agarose gel stained with ethidium 12 bromide, and then, DNA extracts were diluted to a final concentration of approximately 10 ng/μ L. Chipmunk samples were amplified at 12 (EACH01‐12; Anderson et al. ) microsatellite loci, while white‐footed mice were amplified at 10 loci [PO‐26, PO‐85, Po‐97 (Prince et al. ); Pml01, Pml02, Pml05, Pml09, Pml12 (Chirhart et al. ); PLGT15 (Schmidt )]. .. Amplification by multiplex PCR took place in 10 μ L volumes with 20 ng of template DNA, 0.2 mM of each dNTP, 1 U of Taq DNA polymerase (NEB), and 2× Thermopol reaction buffer (20 mM Tris‐HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X‐100; NEB).

    Article Title: Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs
    Article Snippet: PCRs on undamaged DNA For the undamaged plasmid DNA template, 50 µl PCRs were set up containing standard ThermoPol reaction buffer [20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100] (New England Biolabs), 10 ng of circular pJM548 (Ste dpo4 -like in pGEM-T), 200 µM of each ultrapure dNTP and 1 µM of each primer; Ste_FNde5 and Ste_RBam1089 whose sequences are provided above. .. Each of the Dpo4 enzymes was added to a concentration of 200 nM, except the Ssh Dpo4-like enzyme, which was added to a concentration of 400 nM.

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics
    Article Snippet: The primer–template complex was annealed in ThermoPol buffer (1 × : 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8; New England Biolabs) by heating for 5 min at 95 °C and cooling for 5 min at 4 °C. .. The concentration of primer, template and quencher strands were 2 μM, 1 μM and 3 μM, respectively.

    Article Title: Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane
    Article Snippet: Thus, the LAMP reaction conditions in combination 7 were optimal for detecting S. scitamineum in sugarcane, which consisted of Mg2+ at a concentration of 6.00 mmol/L, a ratio of inner vs. outer primers of 6:1, and a concentration of Bst DNA polymerase of 8.0 U. .. The optimized LAMP reaction conditions including 4 mmol/L MgSO4 (50 mmol/L), 1.4 mmol/L dNTPs (10 mmol/L), 2.5 μL 10×ThermoPol Reaction Buffer [containing 20 mmol/L Tris-HCl pH 8.8, 10 mmol/L KCl, 2 mmol/L MgSO4 , 10 mmol/L (NH4 )2 SO4 , 0.1% Triton X-100, NEB company], 1.2 μmol/L each of FIP and BIP primers, 0.2 μmol/L each of F3 and B3 primers, 8.0 U Bst DNA polymerase (8 000 U/mL, NEB company), DNA template 1.0 μL, ddH2 O were complemented to a volume of 25 μL.

    Lamp Assay:

    Article Title: Loop-mediated isothermal amplification (LAMP) assays for the species-specific detection of Eimeria that infect chickens
    Article Snippet: .. LAMP assay Each LAMP reaction was performed in a final volume of 25 μl containing 8 U Bst DNA polymerase (large fragment; New England Biolabs) in 1 × ThermoPol Reaction buffer (20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 and 0.1% Triton X-100; New England Biolabs) supplemented with 2 mM MgCl2 , 1 M betaine and 400 μM of each dNTP. ..

    Article Title: Strong-LAMP: A LAMP Assay for Strongyloides spp. Detection in Stool and Urine Samples. Towards the Diagnosis of Human Strongyloidiasis Starting from a Rodent Model
    Article Snippet: Paragraph title: Optimization of LAMP assay ... Thus, LAMP reactions mixtures (25 μL) contained 40 pmol of each FIP and BIP primers, 5 pmol of each F3 and B3 primers, 1.4 mM of each dNTP (Bioron), 1x ThermoPol Reaction Buffer -20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100; New England Biolabs, UK- (when using Bst Polymerase Large Fragment) or 1x Isothermal Amplification Buffer -20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Tween20; New England Biolabs, UK- (when using either Bst DNA polymerase 2.0 or Bst DNA polymerase 2.0 WarmStart), betaine (ranging 0.8, 1, 1.2, 1.4 or 1.6 M), supplementary MgSO4 (ranging 2, 4, 6 or 8 mM) and 8 U of the tested Bst polymerase in each case with 2 μL of template DNA.

    Marker:

    Article Title: Rapid and sensitive detection of Pseudocercospora eumusae pathogen causing eumusae leaf spot disease of banana by loop-mediated isothermal amplification (LAMP) method
    Article Snippet: The LAMP primers specific to P. eumusae were designed based on the specific SCAR marker sequence of P. eumusae derived from RAPD analysis. .. To determine the optimal reaction temperature and time, the LAMP reaction mixtures were incubated at 56–65 °C for 15, 30, 45, 60, and 90 min. A total volume of 25 ul for the LAMP reaction contained 0.48 µM FIP and BIP, 0.12 µM concentrations of F3 and B3, 2 mM dNTPs mix, 2.0 U of Bst DNA polymerase large fragment (New England Biolabs, Inc., Beverly, MA), 1× ThermoPol buffer (20 mM Tris–HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8) (New England Biolabs), 4 mM MgSO4 and 1 µl of template DNA (25 ng).

    Staining:

    Article Title: Evaluating the influence of life‐history characteristics on genetic structure: a comparison of small mammals inhabiting complex agricultural landscapes
    Article Snippet: DNA quality was checked by running 3 μ L of DNA out on a 2% agarose gel stained with ethidium 12 bromide, and then, DNA extracts were diluted to a final concentration of approximately 10 ng/μ L. Chipmunk samples were amplified at 12 (EACH01‐12; Anderson et al. ) microsatellite loci, while white‐footed mice were amplified at 10 loci [PO‐26, PO‐85, Po‐97 (Prince et al. ); Pml01, Pml02, Pml05, Pml09, Pml12 (Chirhart et al. ); PLGT15 (Schmidt )]. .. Amplification by multiplex PCR took place in 10 μ L volumes with 20 ng of template DNA, 0.2 mM of each dNTP, 1 U of Taq DNA polymerase (NEB), and 2× Thermopol reaction buffer (20 mM Tris‐HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X‐100; NEB).

    Article Title: Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs
    Article Snippet: PCRs on undamaged DNA For the undamaged plasmid DNA template, 50 µl PCRs were set up containing standard ThermoPol reaction buffer [20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 , 0.1% Triton X-100] (New England Biolabs), 10 ng of circular pJM548 (Ste dpo4 -like in pGEM-T), 200 µM of each ultrapure dNTP and 1 µM of each primer; Ste_FNde5 and Ste_RBam1089 whose sequences are provided above. .. Reactions were heated to 85°C for 3 min and then treated with 35 cycles of 85°C for 30 s, 56°C for 1 min and 60°C for 4 min. Products were separated on a 0.9% agarose gel stained with ethidium bromide.

    Article Title: Rapid and sensitive detection of Pseudocercospora eumusae pathogen causing eumusae leaf spot disease of banana by loop-mediated isothermal amplification (LAMP) method
    Article Snippet: To determine the optimal reaction temperature and time, the LAMP reaction mixtures were incubated at 56–65 °C for 15, 30, 45, 60, and 90 min. A total volume of 25 ul for the LAMP reaction contained 0.48 µM FIP and BIP, 0.12 µM concentrations of F3 and B3, 2 mM dNTPs mix, 2.0 U of Bst DNA polymerase large fragment (New England Biolabs, Inc., Beverly, MA), 1× ThermoPol buffer (20 mM Tris–HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8) (New England Biolabs), 4 mM MgSO4 and 1 µl of template DNA (25 ng). .. The visual detection of LAMP products was performed by the addition of 2 µl of 1000× SYBR® Green I nucleic acid gel stain (Invitrogen, Carlsbad, California).

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    New England Biolabs vanadyl ribonucleoside complex
    Vanadyl Ribonucleoside Complex, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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