tris edta buffer  (Thermo Fisher)


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    Name:
    TE Buffer
    Description:
    This 1X TE Buffer is a component of the PureLink 96 Plasmid Purification System now offered separately It is used to resuspend the final purified plasmid pellet and contains very low EDTA so it is compatible with sequencing and other enzymatic applications Composition 10 mM Tris HCl pH 8 0 0 1 mM EDTA
    Catalog Number:
    12090015
    Price:
    None
    Category:
    Lab Reagents and Chemicals
    Applications:
    Automated Plasmid DNA Purification|DNA & RNA Purification & Analysis|DNA Extraction|General Plasmid DNA Purification Reagents & Accessories|Miniprep|Plasmid DNA Purification|Automated Nucleic Acid Purification
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    Structured Review

    Thermo Fisher tris edta buffer
    This 1X TE Buffer is a component of the PureLink 96 Plasmid Purification System now offered separately It is used to resuspend the final purified plasmid pellet and contains very low EDTA so it is compatible with sequencing and other enzymatic applications Composition 10 mM Tris HCl pH 8 0 0 1 mM EDTA
    https://www.bioz.com/result/tris edta buffer/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tris edta buffer - by Bioz Stars, 2021-04
    99/100 stars

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    Related Articles

    Homogenization:

    Article Title: Heavy Metal Neurotoxicants Induce ALS-Linked TDP-43 Pathology
    Article Snippet: A total of ∼60 mg of frozen cortical tissue was lysed by bead mill (Thermo) homogenization in 1 ml Qiazol reagent (Qiagen) then the RNA was extracted following the Lipid tissue RNeasy minikit protocol (Qiagen). qPCR on reverse-transcribed total RNA was performed as described above. .. A total of ∼100 mg of frozen cortical tissue was lysed by bead mill (Thermo) homogenization in Tris-EDTA buffer (10 mM Tris pH8; 1 mM EDTA; 1 mM PMSF; 1× HALT PIC, and PhosSTOP [Roche]). .. Sample concentrations were determined by BCA assay.

    Purification:

    Article Title: MICROBIAL ADHESION OF CRYPTOSPORIDIUM PARVUM SPOROZOITES: PURIFICATION OF AN INHIBITORY LIPID FROM BOVINE MUCOSA
    Article Snippet: Oocysts were purified from collected feces by sequential sieve filtration, Sheather’s sugar floatation, and discontinuous cesium chloride density centrifugation using minor modifications of previously established protocols ( ). .. The final, purified oocysts were washed by centrifugation in Tris–ethylenediamine-tetraacetic acid (Tris-EDTA) buffer (50 mM Tris, 10 mM EDTA) and stored at 4 C in a solution of 50% Hanks’ balanced salt solution (HBSS, GIBCO, Grand Island, New York) and 50% antibiotic–antimycotic solution consisting of 6 g penicillin, 10 g streptomycin, 25 mg amphotericin, and 8.5 g NaCl/L of water. .. Briefly, purified oocysts were washed twice in HBSS by centrifugation at 1,500 g for 15 min, resuspended in HBSS, and treated with an equal volume of 40% (v/v) commercial laundry bleach (5.25% sodium hypochlorite) for 10 min before excystation.

    Centrifugation:

    Article Title: MICROBIAL ADHESION OF CRYPTOSPORIDIUM PARVUM SPOROZOITES: PURIFICATION OF AN INHIBITORY LIPID FROM BOVINE MUCOSA
    Article Snippet: Oocysts were purified from collected feces by sequential sieve filtration, Sheather’s sugar floatation, and discontinuous cesium chloride density centrifugation using minor modifications of previously established protocols ( ). .. The final, purified oocysts were washed by centrifugation in Tris–ethylenediamine-tetraacetic acid (Tris-EDTA) buffer (50 mM Tris, 10 mM EDTA) and stored at 4 C in a solution of 50% Hanks’ balanced salt solution (HBSS, GIBCO, Grand Island, New York) and 50% antibiotic–antimycotic solution consisting of 6 g penicillin, 10 g streptomycin, 25 mg amphotericin, and 8.5 g NaCl/L of water. .. Briefly, purified oocysts were washed twice in HBSS by centrifugation at 1,500 g for 15 min, resuspended in HBSS, and treated with an equal volume of 40% (v/v) commercial laundry bleach (5.25% sodium hypochlorite) for 10 min before excystation.

    Article Title: Identification of Multiple Potent Neutralizing and Non-Neutralizing Antibodies against Epstein-Barr Virus gp350 Protein with Potential for Clinical Application and as Reagents for Mapping Immunodominant Epitopes
    Article Snippet: Puromycin-resistant clones were expanded, followed by flow cytometry sorting using m72A1 to a purity > 90%. .. EBV gp350-positive CHO cells were harvested and lysed in radioimmunoprecipitation assay buffer (RIPA) followed by centrifugation at 15,000 g for 15 min on a benchtop centrifuge. .. The lysate was collected and heated at 95°C for 10 min in SDS sample buffer containing β-mercaptoethanol, then separated using SDS-PAGE.

    Isolation:

    Article Title: Inactivation of group 2 σ factors upregulates production of transcription and translation machineries in the cyanobacterium Synechocystis sp. PCC 6803
    Article Snippet: Total RNAs were isolated from each fraction. .. An aliquot of 300 μL of a fraction was mixed with 200 μL of RNA isolation buffer (20 mM Na-acetate pH 4.5, 2% SDS, 250 mM Na-EDTA) and treated with one unit of DNaseI (Life Technologies) for 5 min and then extracted once with phenol:chlororoform (1:1) and once with chloroform. .. RNAs were precipitated at −20 °C overnight after addition of LiCl to a final concentration of 0.725 M and 2.5 volumes of ethanol.

    Ethanol Precipitation:

    Article Title: Molecular recognition of pyr mRNA by the Bacillus subtilis attenuation regulatory protein PyrR
    Article Snippet: RNA for electrophoretic gel mobility shift assays was produced by in vitro transcription and purified following denaturing acrylamide gel electrophoresis as described by Turner et al . , with the following exceptions. .. In some cases the ethanol precipitation step was replaced by dialyzing the RNA against RNase-free TE buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA) using Slide-A-Lyzer cassettes (Pierce Chemical Co., Rockford, IL), which gave a reproducibly higher yield of RNA. .. Another exception was for the titration experiment to determine the stoichiometry of the PyrR– pyr mRNA interaction, which required a much higher RNA concentration.

    Flow Cytometry:

    Article Title: Characterization of basigin monoclonal antibodies for receptor-mediated drug delivery to the brain
    Article Snippet: .. Flow cytometry analysis The cultured hCMEC/D3 cells were detached with Versene (Gibco Life Technologies, cat#15,040–033), resuspended in flow cytometry buffer (phosphate-buffered saline (PBS), 2% normal goat serum (Jackson ImmunoResearch, cat#005–000-121), 2 mM EDTA), and plated in a 96 well round bottom plate with 1–0.5 × 106 cells/well. .. Cells were stained with Live/dead fixable violet dead cell stain (Thermo Fisher Scientific, cat#L34955) for 15 min and washed twice in flow cytometry buffer before blocking in blocking buffer (PBS, 5% normal goat serum, 2 mM EDTA) for 10 min at 4 °C.

    Cell Culture:

    Article Title: Characterization of basigin monoclonal antibodies for receptor-mediated drug delivery to the brain
    Article Snippet: .. Flow cytometry analysis The cultured hCMEC/D3 cells were detached with Versene (Gibco Life Technologies, cat#15,040–033), resuspended in flow cytometry buffer (phosphate-buffered saline (PBS), 2% normal goat serum (Jackson ImmunoResearch, cat#005–000-121), 2 mM EDTA), and plated in a 96 well round bottom plate with 1–0.5 × 106 cells/well. .. Cells were stained with Live/dead fixable violet dead cell stain (Thermo Fisher Scientific, cat#L34955) for 15 min and washed twice in flow cytometry buffer before blocking in blocking buffer (PBS, 5% normal goat serum, 2 mM EDTA) for 10 min at 4 °C.

    Incubation:

    Article Title: Okinalysin, a Novel P-I Metalloproteinase from Ovophis okinavensis: Biological Properties and Effect on Vascular Endothelial Cells
    Article Snippet: .. The digested fragments were also obtained by autoproteolysis, which occurs when okinalysin is incubated in 10 mM Tris-HCl buffer (pH 7.5) containing 10 mM NaCl at 37 °C for 23 h. The fragments were analyzed by the Edman degradation method using Applied Biosystems 491 protein sequencer and Model 610A PTH analyzer (Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. .. Enzyme Activities and Pharmacological Activities Proteolytic activity was measured by the method of Murata et al. [ ] using casein as the substrate, and arginine ester hydrolytic activity by the method of Roberts [ ].

    Real-time Polymerase Chain Reaction:

    Article Title: Quantitative Polymerase Chain Reaction Analysis of Cariogenic Streptococcus mutans in Saliva of Oral and Laryngeal Cancer Patients Undergoing Radiotherapy: A Clinical Study
    Article Snippet: The collected saliva in the graduated container was measured to check the salivary flow. pH of the saliva was measured using the pH strips (Merck company, Mumbai, India). .. This collected saliva was then transferred to Eppendorf tube containing ethylenediaminetetraacetic acid (EDTA) buffer (TE buffer) (Thermofisher Scientific Inc., Massachusetts, United States) and was sent for real-time PCR analysis (thermal cycler PCR machine-Master cycler, Eppendorf, Germany) [ ]. .. For PCR analysis, Eppendorf tubes containing saliva and TE buffer were centrifuged at 5000 rpm for 5 min and the process was recapitulated for four times.

    Polymerase Chain Reaction:

    Article Title: Quantitative Polymerase Chain Reaction Analysis of Cariogenic Streptococcus mutans in Saliva of Oral and Laryngeal Cancer Patients Undergoing Radiotherapy: A Clinical Study
    Article Snippet: The collected saliva in the graduated container was measured to check the salivary flow. pH of the saliva was measured using the pH strips (Merck company, Mumbai, India). .. This collected saliva was then transferred to Eppendorf tube containing ethylenediaminetetraacetic acid (EDTA) buffer (TE buffer) (Thermofisher Scientific Inc., Massachusetts, United States) and was sent for real-time PCR analysis (thermal cycler PCR machine-Master cycler, Eppendorf, Germany) [ ]. .. For PCR analysis, Eppendorf tubes containing saliva and TE buffer were centrifuged at 5000 rpm for 5 min and the process was recapitulated for four times.

    Radio Immunoprecipitation:

    Article Title: Identification of Multiple Potent Neutralizing and Non-Neutralizing Antibodies against Epstein-Barr Virus gp350 Protein with Potential for Clinical Application and as Reagents for Mapping Immunodominant Epitopes
    Article Snippet: Puromycin-resistant clones were expanded, followed by flow cytometry sorting using m72A1 to a purity > 90%. .. EBV gp350-positive CHO cells were harvested and lysed in radioimmunoprecipitation assay buffer (RIPA) followed by centrifugation at 15,000 g for 15 min on a benchtop centrifuge. .. The lysate was collected and heated at 95°C for 10 min in SDS sample buffer containing β-mercaptoethanol, then separated using SDS-PAGE.

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    Thermo Fisher dye sybr green i
    Loop-mediated isothermal amplification of DNA (LAMP) for detection of M. hapla . ( A ), LAMP products were visually observed with <t>SYBR</t> <t>Green</t> I fluorescence dye. ( B ), 2% agarose Gel electrophoresis separation of LAMP products. ( C ), A lateral flow strips (LFD) detection system was used to detect LAMP amplification. Lines 1–4 represent the M. hapla isolates Mh1, Mh2, Mh3, Mh4, CK, which was the negative control with no template DNA. M represents a DL2000 DNA size marker.
    Dye Sybr Green I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher immunoblot buffer
    Time course for DNA damage and p53 network response in HT1080 cells following exposure to etoposide, quercetin, or methyl methanesulfonate. Representative Western blots from three separate experiments are shown. HT1080 cells were exposed to 0.1% DMSO (control cells; Ctrl), 1-μM etoposide (ETP), 30-μM quercetin (QUE), or 200-μM methyl methanesulfonate (MMS) for 3, 8, or 24 h. Total cellular protein was isolated and subjected to <t>immunoblot</t> analysis. GAPDH was used as an internal control.
    Immunoblot Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher tbe running buffer
    Quantitative AGE assay development. ( A ) Ethidium bromide versus Sybr-Gold ‘Staining’. Aliquots of 200 ng of four plasmid batches known to contain differing levels of recombinant plasmid were separated by AGE and then stained with <t>1×</t> <t>TBE</t> containing either ethidium bromide (50 ng/ml) (left gel) or 1× Sybr-Gold (right gel) for 30 min prior to gel-image capture by Polaroid camera. Arrows indicate the position of low-level recombinants only clearly visible with Sybr-Gold staining. ( B ) Image analysis of a 1× Sybr-Gold stained gel using manufacture's recommended staining times. Image capture by ProXpress. For this analysis, 1, 2 and 5 μl volumes of MassRuler High Range DNA Ladder (MBI-Fermentas, Lithuania) were loaded in lanes 1, 2 and 3, respectively. Left panel, gel image with loads corresponding to DNA quantities (per band) ranging from 1.6 to 10 ng (lane 1), 3.2 to 20 ng (lane 2) and 8 to 50 ng (lane 3). Middle panel, schematic ImageQuant (Molecular Dynamics, SunnyVale, CA) generated electropherogram traces of each lane. Right panel, scatter-plot with trend-line representation of area-under-curve signal ( y -axis) against quantity (in nanograms) of DNA ( x -axis) of the combined 1.6 ng through to 50 ng results. This plot highlights the significant signal ‘plateau’ that develops above 50 ng loads ( C ) Extending assay linear range by increasing Sybr-Gold staining times. Triplicate 5 μl (lanes 1, 3 and 5) and 30 μl (lanes 2, 4 and 6) MassRuler DNA ladder loads were separated by AGE. Subsequently, the gel was then dissected longitudinally into three parts and stained for 20 min (lanes 1 and 2), 2 h (lanes 3 and 4) or 24 h (lanes 5 and 6). After such staining regimes, excess stain was removed by copious washing with water. Image analysis by ProXpress and scatter plot data representation was then undertaken as described above in (B). The 5 and 30 μl loads generate a collective DNA species quantitative range of between 8 and 300 ng. The results of analysis reveal the R 2 values obtained for the 20 min, 2 h and 24 h staining regimes are 0.910, 0.990 and 0.999, respectively.
    Tbe Running Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Loop-mediated isothermal amplification of DNA (LAMP) for detection of M. hapla . ( A ), LAMP products were visually observed with SYBR Green I fluorescence dye. ( B ), 2% agarose Gel electrophoresis separation of LAMP products. ( C ), A lateral flow strips (LFD) detection system was used to detect LAMP amplification. Lines 1–4 represent the M. hapla isolates Mh1, Mh2, Mh3, Mh4, CK, which was the negative control with no template DNA. M represents a DL2000 DNA size marker.

    Journal: Scientific Reports

    Article Title: Rapid, simple and direct detection of Meloidogyne hapla from infected root galls using loop-mediated isothermal amplification combined with FTA technology

    doi: 10.1038/srep44853

    Figure Lengend Snippet: Loop-mediated isothermal amplification of DNA (LAMP) for detection of M. hapla . ( A ), LAMP products were visually observed with SYBR Green I fluorescence dye. ( B ), 2% agarose Gel electrophoresis separation of LAMP products. ( C ), A lateral flow strips (LFD) detection system was used to detect LAMP amplification. Lines 1–4 represent the M. hapla isolates Mh1, Mh2, Mh3, Mh4, CK, which was the negative control with no template DNA. M represents a DL2000 DNA size marker.

    Article Snippet: Analysis of LAMP products The LAMP amplification results were detected with three methods: adding the fluorescent dye SYBR green I (Invitrogen, 1:1000 TE buffer) to the reaction mixture and visually inspecting the results with the naked eye or under UV light; a Lateral-flow dipstick (LFD) assay that as visually observed with naked eyes, and 2% agarose gel electrophoresis.

    Techniques: Amplification, SYBR Green Assay, Fluorescence, Agarose Gel Electrophoresis, Flow Cytometry, Negative Control, Marker

    The sensitivity of the LAMP assay and the conventional PCR for detection of M. hapla . The two methods were carried out at the following, the negative control used water. The conventional PCR was performed with primers F3 and B3. ( A ) Sensitivity of the LAMP products detected by SYBR Green I fluorescence dye ( B ) Sensitivity of the LAMP products detected by gel electrophoresis. ( C ) Sensitivity of the conventional PCR products detected by gel electrophoresis. Concentrations of 10 −0 , 10 −1 , 10 −2 , 10 −3 , 10 −4 , 10 −5 and 10 −6 of single female nematode genomic DNA were used, CK represents no-template control. Lane M represents a DL2000 DNA size marker (ordinate values in bp).

    Journal: Scientific Reports

    Article Title: Rapid, simple and direct detection of Meloidogyne hapla from infected root galls using loop-mediated isothermal amplification combined with FTA technology

    doi: 10.1038/srep44853

    Figure Lengend Snippet: The sensitivity of the LAMP assay and the conventional PCR for detection of M. hapla . The two methods were carried out at the following, the negative control used water. The conventional PCR was performed with primers F3 and B3. ( A ) Sensitivity of the LAMP products detected by SYBR Green I fluorescence dye ( B ) Sensitivity of the LAMP products detected by gel electrophoresis. ( C ) Sensitivity of the conventional PCR products detected by gel electrophoresis. Concentrations of 10 −0 , 10 −1 , 10 −2 , 10 −3 , 10 −4 , 10 −5 and 10 −6 of single female nematode genomic DNA were used, CK represents no-template control. Lane M represents a DL2000 DNA size marker (ordinate values in bp).

    Article Snippet: Analysis of LAMP products The LAMP amplification results were detected with three methods: adding the fluorescent dye SYBR green I (Invitrogen, 1:1000 TE buffer) to the reaction mixture and visually inspecting the results with the naked eye or under UV light; a Lateral-flow dipstick (LFD) assay that as visually observed with naked eyes, and 2% agarose gel electrophoresis.

    Techniques: Lamp Assay, Polymerase Chain Reaction, Negative Control, SYBR Green Assay, Fluorescence, Nucleic Acid Electrophoresis, Marker

    Application of LAMP and conventional PCR on field samples. The number of samples is identical to that in Supplementary Table 1 . N; negative control. P; positive control. ( A ) LAMP products detected by SYBR Green I fluorescence dye ( B ) LAMP products detected by LFD strip. ( C ) Conventional PCR products detected by gel electrophoresis.

    Journal: Scientific Reports

    Article Title: Rapid, simple and direct detection of Meloidogyne hapla from infected root galls using loop-mediated isothermal amplification combined with FTA technology

    doi: 10.1038/srep44853

    Figure Lengend Snippet: Application of LAMP and conventional PCR on field samples. The number of samples is identical to that in Supplementary Table 1 . N; negative control. P; positive control. ( A ) LAMP products detected by SYBR Green I fluorescence dye ( B ) LAMP products detected by LFD strip. ( C ) Conventional PCR products detected by gel electrophoresis.

    Article Snippet: Analysis of LAMP products The LAMP amplification results were detected with three methods: adding the fluorescent dye SYBR green I (Invitrogen, 1:1000 TE buffer) to the reaction mixture and visually inspecting the results with the naked eye or under UV light; a Lateral-flow dipstick (LFD) assay that as visually observed with naked eyes, and 2% agarose gel electrophoresis.

    Techniques: Polymerase Chain Reaction, Negative Control, Positive Control, SYBR Green Assay, Fluorescence, Stripping Membranes, Nucleic Acid Electrophoresis

    Time course for DNA damage and p53 network response in HT1080 cells following exposure to etoposide, quercetin, or methyl methanesulfonate. Representative Western blots from three separate experiments are shown. HT1080 cells were exposed to 0.1% DMSO (control cells; Ctrl), 1-μM etoposide (ETP), 30-μM quercetin (QUE), or 200-μM methyl methanesulfonate (MMS) for 3, 8, or 24 h. Total cellular protein was isolated and subjected to immunoblot analysis. GAPDH was used as an internal control.

    Journal: Toxicological Sciences

    Article Title: Profiling Dose-Dependent Activation of p53-Mediated Signaling Pathways by Chemicals with Distinct Mechanisms of DNA Damage

    doi: 10.1093/toxsci/kfu153

    Figure Lengend Snippet: Time course for DNA damage and p53 network response in HT1080 cells following exposure to etoposide, quercetin, or methyl methanesulfonate. Representative Western blots from three separate experiments are shown. HT1080 cells were exposed to 0.1% DMSO (control cells; Ctrl), 1-μM etoposide (ETP), 30-μM quercetin (QUE), or 200-μM methyl methanesulfonate (MMS) for 3, 8, or 24 h. Total cellular protein was isolated and subjected to immunoblot analysis. GAPDH was used as an internal control.

    Article Snippet: 24 h after plating, cells were exposed to 1-μM etoposide, 30-μM quercetin, or 200-μM methyl methanesulfonate for 3, 8, or 24 h. After chemical treatment, cells were washed twice with PBS, homogenized in immunoblot buffer (0.1-M Tris-HCl pH8.0; 0.05-M ethylenediaminetetraacetic acid, pH 8.0; 0.5 M NaCl; 1% sodium dodecyl sulfate (SDS) and 1% sarkosyl) plus 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail (Pierce).

    Techniques: Western Blot, Isolation

    Quantitative AGE assay development. ( A ) Ethidium bromide versus Sybr-Gold ‘Staining’. Aliquots of 200 ng of four plasmid batches known to contain differing levels of recombinant plasmid were separated by AGE and then stained with 1× TBE containing either ethidium bromide (50 ng/ml) (left gel) or 1× Sybr-Gold (right gel) for 30 min prior to gel-image capture by Polaroid camera. Arrows indicate the position of low-level recombinants only clearly visible with Sybr-Gold staining. ( B ) Image analysis of a 1× Sybr-Gold stained gel using manufacture's recommended staining times. Image capture by ProXpress. For this analysis, 1, 2 and 5 μl volumes of MassRuler High Range DNA Ladder (MBI-Fermentas, Lithuania) were loaded in lanes 1, 2 and 3, respectively. Left panel, gel image with loads corresponding to DNA quantities (per band) ranging from 1.6 to 10 ng (lane 1), 3.2 to 20 ng (lane 2) and 8 to 50 ng (lane 3). Middle panel, schematic ImageQuant (Molecular Dynamics, SunnyVale, CA) generated electropherogram traces of each lane. Right panel, scatter-plot with trend-line representation of area-under-curve signal ( y -axis) against quantity (in nanograms) of DNA ( x -axis) of the combined 1.6 ng through to 50 ng results. This plot highlights the significant signal ‘plateau’ that develops above 50 ng loads ( C ) Extending assay linear range by increasing Sybr-Gold staining times. Triplicate 5 μl (lanes 1, 3 and 5) and 30 μl (lanes 2, 4 and 6) MassRuler DNA ladder loads were separated by AGE. Subsequently, the gel was then dissected longitudinally into three parts and stained for 20 min (lanes 1 and 2), 2 h (lanes 3 and 4) or 24 h (lanes 5 and 6). After such staining regimes, excess stain was removed by copious washing with water. Image analysis by ProXpress and scatter plot data representation was then undertaken as described above in (B). The 5 and 30 μl loads generate a collective DNA species quantitative range of between 8 and 300 ng. The results of analysis reveal the R 2 values obtained for the 20 min, 2 h and 24 h staining regimes are 0.910, 0.990 and 0.999, respectively.

    Journal: Nucleic Acids Research

    Article Title: Effective and robust plasmid topology analysis and the subsequent characterization of the plasmid isoforms thereby observed

    doi: 10.1093/nar/gnh124

    Figure Lengend Snippet: Quantitative AGE assay development. ( A ) Ethidium bromide versus Sybr-Gold ‘Staining’. Aliquots of 200 ng of four plasmid batches known to contain differing levels of recombinant plasmid were separated by AGE and then stained with 1× TBE containing either ethidium bromide (50 ng/ml) (left gel) or 1× Sybr-Gold (right gel) for 30 min prior to gel-image capture by Polaroid camera. Arrows indicate the position of low-level recombinants only clearly visible with Sybr-Gold staining. ( B ) Image analysis of a 1× Sybr-Gold stained gel using manufacture's recommended staining times. Image capture by ProXpress. For this analysis, 1, 2 and 5 μl volumes of MassRuler High Range DNA Ladder (MBI-Fermentas, Lithuania) were loaded in lanes 1, 2 and 3, respectively. Left panel, gel image with loads corresponding to DNA quantities (per band) ranging from 1.6 to 10 ng (lane 1), 3.2 to 20 ng (lane 2) and 8 to 50 ng (lane 3). Middle panel, schematic ImageQuant (Molecular Dynamics, SunnyVale, CA) generated electropherogram traces of each lane. Right panel, scatter-plot with trend-line representation of area-under-curve signal ( y -axis) against quantity (in nanograms) of DNA ( x -axis) of the combined 1.6 ng through to 50 ng results. This plot highlights the significant signal ‘plateau’ that develops above 50 ng loads ( C ) Extending assay linear range by increasing Sybr-Gold staining times. Triplicate 5 μl (lanes 1, 3 and 5) and 30 μl (lanes 2, 4 and 6) MassRuler DNA ladder loads were separated by AGE. Subsequently, the gel was then dissected longitudinally into three parts and stained for 20 min (lanes 1 and 2), 2 h (lanes 3 and 4) or 24 h (lanes 5 and 6). After such staining regimes, excess stain was removed by copious washing with water. Image analysis by ProXpress and scatter plot data representation was then undertaken as described above in (B). The 5 and 30 μl loads generate a collective DNA species quantitative range of between 8 and 300 ng. The results of analysis reveal the R 2 values obtained for the 20 min, 2 h and 24 h staining regimes are 0.910, 0.990 and 0.999, respectively.

    Article Snippet: The 1× TBE running buffer also contained 5.0 μg/ml chloroquine, and electrophoresis was performed at 50 V for 48 h. Staining was carried out by soaking in 1× TBE containing 1× Sybr-Gold (Molecular Probes) for 24 h in the dark.

    Techniques: Staining, Plasmid Preparation, Recombinant, Generated