Structured Review

Biotium evagreen
Reverse transcriptase assays. A. Fluorogenic assay. RT activity was measured by detection of RNA:DNA heteroduplex by fluorescence of <t>EvaGreen</t> binding. Oligo dT primed poly A was incubated at 37°C and 65°C in the presence of indicated Pol enzymes in manufacturer recommended buffers and dTTP. Fluorescence measurements were obtained every 6 seconds for 10 minutes. The initial slopes from a plot of RFU vs. time in seconds were determined by linear least square regression from 30 to 150 seconds at 37°C and from 30 to 90 seconds at 65°C. Error bars are standard error of regression slope. B. RT primer extension assay. HEX-labeled dT 20 primed poly A was incubated 10 minutes at 37°C and then 10 minutes at 65°C in the presence of indicated Pol enzymes and dTTP in manufacturer recommended buffers. Primer extension products were resolved by 10% denaturing PAGE and imaged on a Molecular Imager FX (Bio-Rad). Left facing triangle indicates migration of unextended dT20 primer and asterisk indicates bromophenol blue dye front. C. RT MS2-specific primer extension. 5′-labeled primer was annealed to MS2 RNA and incubated 10 minutes at 37°C and then 30 minutes at 65°C in the presence of indicated Pol enzymes with dNTPS (N = A,C,G,T) in manufacturer recommended buffers. Primer extension products were resolved by 5% denaturing PAGE. Lane 1 No RNA+MMLV RT; Lane 2: MS2 RNA No RT; Lane 3 MS2 RNA+MMLV RT, Lane 3 MS2 RNA+3173 Pol. Molecular weight in bases indicted. Red Arrow: ∼650 base MMLV extension product. Blue Arrow: ∼715 base PyroScript extension product. Green arrow: Non-templated MMLV reaction product.
Evagreen, supplied by Biotium, used in various techniques. Bioz Stars score: 94/100, based on 124 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme"

Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme

Journal: PLoS ONE

doi: 10.1371/journal.pone.0038371

Reverse transcriptase assays. A. Fluorogenic assay. RT activity was measured by detection of RNA:DNA heteroduplex by fluorescence of EvaGreen binding. Oligo dT primed poly A was incubated at 37°C and 65°C in the presence of indicated Pol enzymes in manufacturer recommended buffers and dTTP. Fluorescence measurements were obtained every 6 seconds for 10 minutes. The initial slopes from a plot of RFU vs. time in seconds were determined by linear least square regression from 30 to 150 seconds at 37°C and from 30 to 90 seconds at 65°C. Error bars are standard error of regression slope. B. RT primer extension assay. HEX-labeled dT 20 primed poly A was incubated 10 minutes at 37°C and then 10 minutes at 65°C in the presence of indicated Pol enzymes and dTTP in manufacturer recommended buffers. Primer extension products were resolved by 10% denaturing PAGE and imaged on a Molecular Imager FX (Bio-Rad). Left facing triangle indicates migration of unextended dT20 primer and asterisk indicates bromophenol blue dye front. C. RT MS2-specific primer extension. 5′-labeled primer was annealed to MS2 RNA and incubated 10 minutes at 37°C and then 30 minutes at 65°C in the presence of indicated Pol enzymes with dNTPS (N = A,C,G,T) in manufacturer recommended buffers. Primer extension products were resolved by 5% denaturing PAGE. Lane 1 No RNA+MMLV RT; Lane 2: MS2 RNA No RT; Lane 3 MS2 RNA+MMLV RT, Lane 3 MS2 RNA+3173 Pol. Molecular weight in bases indicted. Red Arrow: ∼650 base MMLV extension product. Blue Arrow: ∼715 base PyroScript extension product. Green arrow: Non-templated MMLV reaction product.
Figure Legend Snippet: Reverse transcriptase assays. A. Fluorogenic assay. RT activity was measured by detection of RNA:DNA heteroduplex by fluorescence of EvaGreen binding. Oligo dT primed poly A was incubated at 37°C and 65°C in the presence of indicated Pol enzymes in manufacturer recommended buffers and dTTP. Fluorescence measurements were obtained every 6 seconds for 10 minutes. The initial slopes from a plot of RFU vs. time in seconds were determined by linear least square regression from 30 to 150 seconds at 37°C and from 30 to 90 seconds at 65°C. Error bars are standard error of regression slope. B. RT primer extension assay. HEX-labeled dT 20 primed poly A was incubated 10 minutes at 37°C and then 10 minutes at 65°C in the presence of indicated Pol enzymes and dTTP in manufacturer recommended buffers. Primer extension products were resolved by 10% denaturing PAGE and imaged on a Molecular Imager FX (Bio-Rad). Left facing triangle indicates migration of unextended dT20 primer and asterisk indicates bromophenol blue dye front. C. RT MS2-specific primer extension. 5′-labeled primer was annealed to MS2 RNA and incubated 10 minutes at 37°C and then 30 minutes at 65°C in the presence of indicated Pol enzymes with dNTPS (N = A,C,G,T) in manufacturer recommended buffers. Primer extension products were resolved by 5% denaturing PAGE. Lane 1 No RNA+MMLV RT; Lane 2: MS2 RNA No RT; Lane 3 MS2 RNA+MMLV RT, Lane 3 MS2 RNA+3173 Pol. Molecular weight in bases indicted. Red Arrow: ∼650 base MMLV extension product. Blue Arrow: ∼715 base PyroScript extension product. Green arrow: Non-templated MMLV reaction product.

Techniques Used: Activity Assay, Fluorescence, Binding Assay, Incubation, Primer Extension Assay, Labeling, Polyacrylamide Gel Electrophoresis, Migration, Molecular Weight

2) Product Images from "A rapid, low-cost, and microfluidic chip-based system for parallel identification of multiple pathogens related to clinical pneumonia"

Article Title: A rapid, low-cost, and microfluidic chip-based system for parallel identification of multiple pathogens related to clinical pneumonia

Journal: Scientific Reports

doi: 10.1038/s41598-017-06739-2

Structure of the microfluidic chip and method for the parallel identification of multiple pathogens. ( A ) Basement of the microfluidic chip. ( B ) Cover of the microfluidic chip. ( C ) Six primers embedded together at the bottom of one test cell using low melting point Sepharose CL-4B. ( D ) The mixture of the prepared DNA sample and isothermal nucleic acid amplification reactants is injected into the microfluidic chip via the inlet hole using a pipette. ( E ) The mixtures after being centrifuged at 5000 rpm. ( F ) Six primers released at > 50 °C. ( G ) The fluorescent marker EvaGreen bound to the amplified products as nucleic acid amplification occurred at 65 °C.
Figure Legend Snippet: Structure of the microfluidic chip and method for the parallel identification of multiple pathogens. ( A ) Basement of the microfluidic chip. ( B ) Cover of the microfluidic chip. ( C ) Six primers embedded together at the bottom of one test cell using low melting point Sepharose CL-4B. ( D ) The mixture of the prepared DNA sample and isothermal nucleic acid amplification reactants is injected into the microfluidic chip via the inlet hole using a pipette. ( E ) The mixtures after being centrifuged at 5000 rpm. ( F ) Six primers released at > 50 °C. ( G ) The fluorescent marker EvaGreen bound to the amplified products as nucleic acid amplification occurred at 65 °C.

Techniques Used: Chromatin Immunoprecipitation, Amplification, Injection, Transferring, Marker

3) Product Images from "A high-throughput qPCR system for simultaneous quantitative detection of dairy Lactococcus lactis and Leuconostoc bacteriophages"

Article Title: A high-throughput qPCR system for simultaneous quantitative detection of dairy Lactococcus lactis and Leuconostoc bacteriophages

Journal: PLoS ONE

doi: 10.1371/journal.pone.0174223

Performance of SYBR Green I and EvaGreen detection chemistries during qPCR assays. (A) Standard curves generated from amplification of serially diluted L . lactis phage P220 genome detected with the corresponding chemistries; (B) the corresponding performance parameters; and (C) amplification plots detected with SYBR Green I (brown) and EvaGreen (green).
Figure Legend Snippet: Performance of SYBR Green I and EvaGreen detection chemistries during qPCR assays. (A) Standard curves generated from amplification of serially diluted L . lactis phage P220 genome detected with the corresponding chemistries; (B) the corresponding performance parameters; and (C) amplification plots detected with SYBR Green I (brown) and EvaGreen (green).

Techniques Used: SYBR Green Assay, Real-time Polymerase Chain Reaction, Generated, Amplification

4) Product Images from "Mechanism of heat stress-induced cellular senescence elucidates the exclusive vulnerability of early S-phase cells to mild genotoxic stress"

Article Title: Mechanism of heat stress-induced cellular senescence elucidates the exclusive vulnerability of early S-phase cells to mild genotoxic stress

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv573

Early S-phase cells undergo senescence-like proliferation arrest in response to HS. ( A and B ) Human HeLa cells that were untreated or treated with HS (45.5°C, 30 min) were allowed to recover for 72 h and then either pulse-labelled with EdU (A; 10 μM, 30 min) or stained for SA-β-gal activity (B). EdU was revealed by Click Chemistry (red). The DNA was stained with DAPI (blue). Cells with enlarged nuclei are indicated by white arrows or circles. Scale bar: 25 μm. ( C ) Human HeLa cells that were either untreated, treated with HS (45.5°C, 30 min), or treated with HS and allowed to recover for the indicated time intervals (6, 24 and 72 h) were subjected to gene expression analysis using qRT-PCR and WB. The expression of p21 CIP1 and p16 INK4a was analysed using EvaGreen-based qRT-PCR. The amplification levels of the cDNA were normalised to the level of GAPDH cDNA. The data are represented as the mean ± SEM. WB was carried out with an antibody against p21; histone H3 was used as the loading control. ( D ) Experimental design for comparison of the effects HS on HeLa cells at different cell cycle phases. ( E ) Cell cycle profiles of the cells treated and allowed to recover as in (D). ( F ) Early and late S-phase HeLa cells obtained using a double-thymidine block were heat treated (45.5°C, 30 min), allowed to recover for 72 h and either pulse-labelled with EdU (10 μM, 30 min) or stained for cyclin B1, p21 or SA-β-gal activity. EdU was revealed by Click Chemistry (red). The DNA was stained with DAPI (blue). The control represents HeLa cells synchronised by a double-thymidine block and released for 72 h. Scale bar: 25 μm.
Figure Legend Snippet: Early S-phase cells undergo senescence-like proliferation arrest in response to HS. ( A and B ) Human HeLa cells that were untreated or treated with HS (45.5°C, 30 min) were allowed to recover for 72 h and then either pulse-labelled with EdU (A; 10 μM, 30 min) or stained for SA-β-gal activity (B). EdU was revealed by Click Chemistry (red). The DNA was stained with DAPI (blue). Cells with enlarged nuclei are indicated by white arrows or circles. Scale bar: 25 μm. ( C ) Human HeLa cells that were either untreated, treated with HS (45.5°C, 30 min), or treated with HS and allowed to recover for the indicated time intervals (6, 24 and 72 h) were subjected to gene expression analysis using qRT-PCR and WB. The expression of p21 CIP1 and p16 INK4a was analysed using EvaGreen-based qRT-PCR. The amplification levels of the cDNA were normalised to the level of GAPDH cDNA. The data are represented as the mean ± SEM. WB was carried out with an antibody against p21; histone H3 was used as the loading control. ( D ) Experimental design for comparison of the effects HS on HeLa cells at different cell cycle phases. ( E ) Cell cycle profiles of the cells treated and allowed to recover as in (D). ( F ) Early and late S-phase HeLa cells obtained using a double-thymidine block were heat treated (45.5°C, 30 min), allowed to recover for 72 h and either pulse-labelled with EdU (10 μM, 30 min) or stained for cyclin B1, p21 or SA-β-gal activity. EdU was revealed by Click Chemistry (red). The DNA was stained with DAPI (blue). The control represents HeLa cells synchronised by a double-thymidine block and released for 72 h. Scale bar: 25 μm.

Techniques Used: Staining, Activity Assay, Expressing, Quantitative RT-PCR, Western Blot, Amplification, Blocking Assay

5) Product Images from "One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV"

Article Title: One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2016.02166

EvaGreen activity for RT-LAMP. Resultant microchamber devices following the loop-mediated isothermal amplification (LAMP) reaction with EvaGreen (5 and 10X) under UV (On). DNA ladder-like LAMP amplification pattern was confirmed by 2% agarose gel electrophoresis. T: template; P: LAMP-primers. The fluorescence backgrounds were shown as red-squares.
Figure Legend Snippet: EvaGreen activity for RT-LAMP. Resultant microchamber devices following the loop-mediated isothermal amplification (LAMP) reaction with EvaGreen (5 and 10X) under UV (On). DNA ladder-like LAMP amplification pattern was confirmed by 2% agarose gel electrophoresis. T: template; P: LAMP-primers. The fluorescence backgrounds were shown as red-squares.

Techniques Used: Activity Assay, Amplification, Agarose Gel Electrophoresis, Fluorescence

Sensitivity of one-pot RT-LAMP. (A) 10X EvaGreen was initially mixed with RT-LAMP reagents, and the reaction was performed and visualized in microchamber. The purified MERS-CoV RNA was serially diluted in the range 4 × 10 3 –4 × 10 -1 /μL. Relative fluorescent signals were analyzed by using ImageJ software. The relative intensity of fluorescence for positive amplification was normalized to the controls (-Template/-Primer). Red square indicates background fluorescence. (B) Agarose gel electrophoresis confirmation indicated that one-pot RT-LAMP can detect MERS-CoV as few as 0.4 RNA copies. DNA ladder-like pattern was confirmed by 2% agarose gel electrophoresis. T, template; P, primer.
Figure Legend Snippet: Sensitivity of one-pot RT-LAMP. (A) 10X EvaGreen was initially mixed with RT-LAMP reagents, and the reaction was performed and visualized in microchamber. The purified MERS-CoV RNA was serially diluted in the range 4 × 10 3 –4 × 10 -1 /μL. Relative fluorescent signals were analyzed by using ImageJ software. The relative intensity of fluorescence for positive amplification was normalized to the controls (-Template/-Primer). Red square indicates background fluorescence. (B) Agarose gel electrophoresis confirmation indicated that one-pot RT-LAMP can detect MERS-CoV as few as 0.4 RNA copies. DNA ladder-like pattern was confirmed by 2% agarose gel electrophoresis. T, template; P, primer.

Techniques Used: Purification, Software, Fluorescence, Amplification, Agarose Gel Electrophoresis

Specificity of one-pot RT-LAMP for MERS-CoV. RNA samples of MERS-CoV and the other respiratory pathogens were used for specificity evaluation of one-pot RT-LAMP system for MERS-CoV detection; fluorescence was visualized by 10X EvaGreen in microchamber. Each set of lane indicates a specific virus: lane 1, MERS-CoV Korean isolate; lane 2, Human coronavirus (HCoV)-229E; lane 3, Human metapneumovirus (HMPV); lane 4, B/Brisbane/60/2008 (Victoria lineage); lane 5, B/Phuket/3073/2013 (Yamagata lineage); lane 6, Influenza A virus (A/California/04/2009, H1N1); lane 7, influenza A virus (A/Perth/16/2009, H3N2). M, 100 bp DNA ladder.
Figure Legend Snippet: Specificity of one-pot RT-LAMP for MERS-CoV. RNA samples of MERS-CoV and the other respiratory pathogens were used for specificity evaluation of one-pot RT-LAMP system for MERS-CoV detection; fluorescence was visualized by 10X EvaGreen in microchamber. Each set of lane indicates a specific virus: lane 1, MERS-CoV Korean isolate; lane 2, Human coronavirus (HCoV)-229E; lane 3, Human metapneumovirus (HMPV); lane 4, B/Brisbane/60/2008 (Victoria lineage); lane 5, B/Phuket/3073/2013 (Yamagata lineage); lane 6, Influenza A virus (A/California/04/2009, H1N1); lane 7, influenza A virus (A/Perth/16/2009, H3N2). M, 100 bp DNA ladder.

Techniques Used: Fluorescence

6) Product Images from "Transformation of Personal Computers and Mobile Phones into Genetic Diagnostic Systems"

Article Title: Transformation of Personal Computers and Mobile Phones into Genetic Diagnostic Systems

Journal: Analytical Chemistry

doi: 10.1021/ac5022419

Validation of DNA amplicon detection using a mobile phone camera. (a) Samples containing a range of template molecules were amplified for 30 cycles with EvaGreen, excited by UV transillumination, and imaged with a mobile phone camera and 520 nm filter. (b) Sensitivity and specificity of camera phone detection of amplified DNA in comparison to qPCR end-point detection. The normalized fluorescence for 10 independent experiments is plotted versus the log of the initial template mass.
Figure Legend Snippet: Validation of DNA amplicon detection using a mobile phone camera. (a) Samples containing a range of template molecules were amplified for 30 cycles with EvaGreen, excited by UV transillumination, and imaged with a mobile phone camera and 520 nm filter. (b) Sensitivity and specificity of camera phone detection of amplified DNA in comparison to qPCR end-point detection. The normalized fluorescence for 10 independent experiments is plotted versus the log of the initial template mass.

Techniques Used: Amplification, Real-time Polymerase Chain Reaction, Fluorescence

7) Product Images from "Efficiency of whole genome amplification of single circulating tumor cells enriched by CellSearch and sorted by FACS"

Article Title: Efficiency of whole genome amplification of single circulating tumor cells enriched by CellSearch and sorted by FACS

Journal: Genome Medicine

doi: 10.1186/gm510

Flow-cytometric analysis of CellSearch-enriched circulating tumor cells and real-time whole genome amplification of single-sorted leukocytes and circulating tumor cells. Panels (A) and (B) show the sort gates to identify and sort single CTC, single leukocytes and beads. Panels (C) (Table 1 , patient 7), (E) (Table 1 , patient 1) and (G) (Table 1 , patient 2) show the analysis and sort gates of three patients with lung cancer. Panels (D) , (F) and (H) show the corresponding real-time DNA amplification of the individual sorted cells. The curves show the Evagreen fluorescence of the whole genome amplification reaction mixes in time. APC = allophycocyanin, PE = phycoerythrin, SSC = sidescatter.
Figure Legend Snippet: Flow-cytometric analysis of CellSearch-enriched circulating tumor cells and real-time whole genome amplification of single-sorted leukocytes and circulating tumor cells. Panels (A) and (B) show the sort gates to identify and sort single CTC, single leukocytes and beads. Panels (C) (Table 1 , patient 7), (E) (Table 1 , patient 1) and (G) (Table 1 , patient 2) show the analysis and sort gates of three patients with lung cancer. Panels (D) , (F) and (H) show the corresponding real-time DNA amplification of the individual sorted cells. The curves show the Evagreen fluorescence of the whole genome amplification reaction mixes in time. APC = allophycocyanin, PE = phycoerythrin, SSC = sidescatter.

Techniques Used: Flow Cytometry, Whole Genome Amplification, Amplification, Fluorescence

Yields of each of the steps for whole genome amplification. SKBR-3 cells were sorted into aliquots of 7.5 ml of blood containing 500, 50 and 5 cells, and enriched and enumerated by CellSearch. The contents of the CellSearch cartridge was placed in a FACS tube and SKBR-3 cells sorted into a 384-well plate. On each well, a GE GenomiPhi amplification reaction was performed in the presence of Evagreen. The yield of each step in the procedure was determined and plotted as a percentage of the starting amount. Each experiment was performed in triplicate except for the 5-cell sort, which was done six times. FACS, fluorescence-activated cell sorting.
Figure Legend Snippet: Yields of each of the steps for whole genome amplification. SKBR-3 cells were sorted into aliquots of 7.5 ml of blood containing 500, 50 and 5 cells, and enriched and enumerated by CellSearch. The contents of the CellSearch cartridge was placed in a FACS tube and SKBR-3 cells sorted into a 384-well plate. On each well, a GE GenomiPhi amplification reaction was performed in the presence of Evagreen. The yield of each step in the procedure was determined and plotted as a percentage of the starting amount. Each experiment was performed in triplicate except for the 5-cell sort, which was done six times. FACS, fluorescence-activated cell sorting.

Techniques Used: Whole Genome Amplification, FACS, Amplification, Fluorescence

8) Product Images from "Inducing cellular senescence in vitro by using genetically encoded photosensitizers"

Article Title: Inducing cellular senescence in vitro by using genetically encoded photosensitizers

Journal: Aging (Albany NY)

doi: 10.18632/aging.101065

Analysis of the expression of p21 and p16 CDK inhibitors in HeLa cells expressing genetically encoded photosensitizers HeLa cells that express H2B-miniSOG or H2B-tKR were synchronized in S phase, illuminated with blue (465-495 nm, 65 mW/cm 2 , 5 min) or green (540-580 nm, 200 mW/cm 2 , 15 min) light, allowed to recover for the indicated time intervals (0, 6, 24 and 48 hr), and subjected to gene expression analysis using qRT-PCR and WB. Control (“C”) represents the non-illuminated cells. The expression of p21 CIP1 and p16 INK4a was analyzed using EvaGreen-based qRT-PCR. The amplification levels of the cDNA were normalized to the level of the GAPDH cDNA. The results of one representative experiment are shown. WB was performed with an antibody against p21; GAPDH was used as the loading control.
Figure Legend Snippet: Analysis of the expression of p21 and p16 CDK inhibitors in HeLa cells expressing genetically encoded photosensitizers HeLa cells that express H2B-miniSOG or H2B-tKR were synchronized in S phase, illuminated with blue (465-495 nm, 65 mW/cm 2 , 5 min) or green (540-580 nm, 200 mW/cm 2 , 15 min) light, allowed to recover for the indicated time intervals (0, 6, 24 and 48 hr), and subjected to gene expression analysis using qRT-PCR and WB. Control (“C”) represents the non-illuminated cells. The expression of p21 CIP1 and p16 INK4a was analyzed using EvaGreen-based qRT-PCR. The amplification levels of the cDNA were normalized to the level of the GAPDH cDNA. The results of one representative experiment are shown. WB was performed with an antibody against p21; GAPDH was used as the loading control.

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Amplification

9) Product Images from "Quantification of Mitochondrial DNA (mtDNA) Damage and Error Rates by Real-time QPCR"

Article Title: Quantification of Mitochondrial DNA (mtDNA) Damage and Error Rates by Real-time QPCR

Journal: Mitochondrion

doi: 10.1016/j.mito.2008.11.004

LRCPR of mtDNA using increasing concentrations of EvaGreen. A. Crossing point analysis of the LRPCR using increasing concentrations of EvaGreen. 100ng of total DNA was used as described in Methods. B. 10% of the LRPCR product after 40 cycles of amplification was electrophoresed on a 1% agarose gel. The integrated density value (IDV) of the bands was determined using AlphaInnotech/AlphaEaseFC software following 40 cycles of amplification.
Figure Legend Snippet: LRCPR of mtDNA using increasing concentrations of EvaGreen. A. Crossing point analysis of the LRPCR using increasing concentrations of EvaGreen. 100ng of total DNA was used as described in Methods. B. 10% of the LRPCR product after 40 cycles of amplification was electrophoresed on a 1% agarose gel. The integrated density value (IDV) of the bands was determined using AlphaInnotech/AlphaEaseFC software following 40 cycles of amplification.

Techniques Used: Amplification, Agarose Gel Electrophoresis, Software

Comparison of realtime LRPCR and step cycle amplification of mtDNA. A. Representative picture of step LRPCR; 100 ng of total DNA was amplified using the LRPCR protocol and the reaction tube was removed at the cycle number indicated above each band. 10% of the LRPCR product was run on a 1% agarose gel. B. Realtime determination of DNA fluorescence (n=4) was determined as described in Methods. For the step cycle, the integrated density value (IDV) of the bands (n=3 for each step) was determined using AlphaInnotech/AlphaEaseFC software. All individual reactions contained 1:1250 dilution of EvaGreen. Values represented are mean±SEM of fluorescence normalized to the plateau values.
Figure Legend Snippet: Comparison of realtime LRPCR and step cycle amplification of mtDNA. A. Representative picture of step LRPCR; 100 ng of total DNA was amplified using the LRPCR protocol and the reaction tube was removed at the cycle number indicated above each band. 10% of the LRPCR product was run on a 1% agarose gel. B. Realtime determination of DNA fluorescence (n=4) was determined as described in Methods. For the step cycle, the integrated density value (IDV) of the bands (n=3 for each step) was determined using AlphaInnotech/AlphaEaseFC software. All individual reactions contained 1:1250 dilution of EvaGreen. Values represented are mean±SEM of fluorescence normalized to the plateau values.

Techniques Used: Amplification, Agarose Gel Electrophoresis, Fluorescence, Software

10) Product Images from "Melting Temperature Mapping Method: A Novel Method for Rapid Identification of Unknown Pathogenic Microorganisms within Three Hours of Sample Collection"

Article Title: Melting Temperature Mapping Method: A Novel Method for Rapid Identification of Unknown Pathogenic Microorganisms within Three Hours of Sample Collection

Journal: Scientific Reports

doi: 10.1038/srep12543

Concept of the Tm mapping method. ( A ) The strategy for the primer designs is shown. Nested PCR is performed using seven bacterial universal primer sets, and then the seven Tm values are obtained. ( B ) Mapping the seven Tm values on two dimensions leads to the identification of the unique bacterial species-specific shape. The average of all seven Tm values includes the measurement error among trials; however, the Tm mapping shape is not affected by this type of error. ( C ) Using an analytical instrument with a high degree of thermal accuracy among PCR tubes and Tm value analysis with EvaGreen dye in 36 samples of the same bacterial DNA in the same trial, the tube-to-tube variation is within ±0.1 °C. ( D ) In order to analyze the Tm mapping “shape”, we developed a method to measure the distance of each individual Tm value from the average value. Tm values above the average receive a “+” designation, while those below the average receive a “−” designation. The Tm mapping shape is identified by comparing the seven distances obtained from the unknown bacteria to those in the database. ( E ) In order to identify a bacterial isolate, the identification software program calculates the Difference Values using the indicated formula. The closer the Difference Value is to zero, the more similar the Tm mapping shape is to the shape of a given species of pathogenic bacteria in the database.
Figure Legend Snippet: Concept of the Tm mapping method. ( A ) The strategy for the primer designs is shown. Nested PCR is performed using seven bacterial universal primer sets, and then the seven Tm values are obtained. ( B ) Mapping the seven Tm values on two dimensions leads to the identification of the unique bacterial species-specific shape. The average of all seven Tm values includes the measurement error among trials; however, the Tm mapping shape is not affected by this type of error. ( C ) Using an analytical instrument with a high degree of thermal accuracy among PCR tubes and Tm value analysis with EvaGreen dye in 36 samples of the same bacterial DNA in the same trial, the tube-to-tube variation is within ±0.1 °C. ( D ) In order to analyze the Tm mapping “shape”, we developed a method to measure the distance of each individual Tm value from the average value. Tm values above the average receive a “+” designation, while those below the average receive a “−” designation. The Tm mapping shape is identified by comparing the seven distances obtained from the unknown bacteria to those in the database. ( E ) In order to identify a bacterial isolate, the identification software program calculates the Difference Values using the indicated formula. The closer the Difference Value is to zero, the more similar the Tm mapping shape is to the shape of a given species of pathogenic bacteria in the database.

Techniques Used: Nested PCR, Polymerase Chain Reaction, Software

11) Product Images from "Recombinase‐Based Isothermal Amplification of Nucleic Acids with Self‐Avoiding Molecular Recognition Systems (SAMRS)"

Article Title: Recombinase‐Based Isothermal Amplification of Nucleic Acids with Self‐Avoiding Molecular Recognition Systems (SAMRS)

Journal: Chembiochem

doi: 10.1002/cbic.201402250

RPA with SAMRS primers. A) Native PAGE of RPA products generated by using primers for rpoB containing standard nucleotides (STD) or primers containing standard and SAMRS nucleotides (SAMRS). Lane 1: water; lane 2: 10 2 copies; lane 3: 10 3 copies; lane 4: 10 4 copies; lane 5: 10 5 copies of a plasmid coding for RpoB served as templates in these reactions. The expected size of the product was 102 base‐pairs. PAGE analysis clearly shows the noise in the amplified products with STD primers (A, bottom left) versus SAMRS primers (A, bottom right). B) Real‐time RPA using STD (left) and SAMRS primers (right) for rpoB gene. Fluorescence upon binding of EvaGreen to the amplified product was detected over 50 min. DNA template: 0 copies (blue), 10 copies (purple), 10 2 copies (gray), 10 3 copies (orange), 10 4 copies (red). Insets: correlation between C p ( y ‐axis, time in minutes) and the logarithm of the input template copy number ( x ‐axis).
Figure Legend Snippet: RPA with SAMRS primers. A) Native PAGE of RPA products generated by using primers for rpoB containing standard nucleotides (STD) or primers containing standard and SAMRS nucleotides (SAMRS). Lane 1: water; lane 2: 10 2 copies; lane 3: 10 3 copies; lane 4: 10 4 copies; lane 5: 10 5 copies of a plasmid coding for RpoB served as templates in these reactions. The expected size of the product was 102 base‐pairs. PAGE analysis clearly shows the noise in the amplified products with STD primers (A, bottom left) versus SAMRS primers (A, bottom right). B) Real‐time RPA using STD (left) and SAMRS primers (right) for rpoB gene. Fluorescence upon binding of EvaGreen to the amplified product was detected over 50 min. DNA template: 0 copies (blue), 10 copies (purple), 10 2 copies (gray), 10 3 copies (orange), 10 4 copies (red). Insets: correlation between C p ( y ‐axis, time in minutes) and the logarithm of the input template copy number ( x ‐axis).

Techniques Used: Recombinase Polymerase Amplification, Clear Native PAGE, Generated, Plasmid Preparation, Polyacrylamide Gel Electrophoresis, Amplification, Fluorescence, Binding Assay

Reverse transcription and RPA (RT‐RPA) with SAMRS primers of a small region in ORF1a of MERS viral RNA. A) Native PAGE of RPA products generated by using primers for MERS with STD primers or SAMRS primers. Lane 1: water; lane 2: 1 pfu; lane 3: 10 pfu; lane 4: 10 2 pfu; lane 5: 10 3 pfu of MERS viral RNA served as templates in these reactions. The expected size of the product was 122 base‐pairs. PAGE analysis clearly depicts primer‐dimers and other artifacts in the amplified products with STD primers (A, bottom left) but not in the assay with SAMRS primers (A, bottom right). B) Real‐time RPA using STD (left) and SAMRS primers (right) for MERS viral RNA. Fluorescence upon binding of EvaGreen to amplified product was detected over 60 min. MERS viral RNA template: 0 pfu (blue), 10 pfu (purple), 10 2 pfu (gray), 10 3 pfu (orange), 10 4 pfu (red). Insets: correlation between C p ( y ‐axis, time in minutes) and the logarithm of the input template copy number ( x ‐axis).
Figure Legend Snippet: Reverse transcription and RPA (RT‐RPA) with SAMRS primers of a small region in ORF1a of MERS viral RNA. A) Native PAGE of RPA products generated by using primers for MERS with STD primers or SAMRS primers. Lane 1: water; lane 2: 1 pfu; lane 3: 10 pfu; lane 4: 10 2 pfu; lane 5: 10 3 pfu of MERS viral RNA served as templates in these reactions. The expected size of the product was 122 base‐pairs. PAGE analysis clearly depicts primer‐dimers and other artifacts in the amplified products with STD primers (A, bottom left) but not in the assay with SAMRS primers (A, bottom right). B) Real‐time RPA using STD (left) and SAMRS primers (right) for MERS viral RNA. Fluorescence upon binding of EvaGreen to amplified product was detected over 60 min. MERS viral RNA template: 0 pfu (blue), 10 pfu (purple), 10 2 pfu (gray), 10 3 pfu (orange), 10 4 pfu (red). Insets: correlation between C p ( y ‐axis, time in minutes) and the logarithm of the input template copy number ( x ‐axis).

Techniques Used: Recombinase Polymerase Amplification, Clear Native PAGE, Generated, Polyacrylamide Gel Electrophoresis, Amplification, Fluorescence, Binding Assay

12) Product Images from "Simple and robust diagnosis of early, small and AFP-negative primary hepatic carcinomas: an integrative approach of serum fluorescence and conventional blood tests"

Article Title: Simple and robust diagnosis of early, small and AFP-negative primary hepatic carcinomas: an integrative approach of serum fluorescence and conventional blood tests

Journal: Oncotarget

doi: 10.18632/oncotarget.11771

Diagram of the measurement of serum autofluorescence and cell-free DNA-related fluorescence FS3T8, FS15T8, FS3T37, FS15T37, FS3T8E, FS15T8E, FS3T37E and FS15T37E: the names of 8 original fluorescence indicators. Each indicator name is an abbreviation indicating the fluorescence intensity (F) of serum (S) 3 μL (3) or 15 μL (15) at a given temperature (T) 8°C (8) or 37°C (37) in the presence (E) or absence of EvaGreen.
Figure Legend Snippet: Diagram of the measurement of serum autofluorescence and cell-free DNA-related fluorescence FS3T8, FS15T8, FS3T37, FS15T37, FS3T8E, FS15T8E, FS3T37E and FS15T37E: the names of 8 original fluorescence indicators. Each indicator name is an abbreviation indicating the fluorescence intensity (F) of serum (S) 3 μL (3) or 15 μL (15) at a given temperature (T) 8°C (8) or 37°C (37) in the presence (E) or absence of EvaGreen.

Techniques Used: Fluorescence

Optimum conditions for the fluorescence intensity measurements of pooled serum samples ( A ) The fluorescence intensity ratios of PHC to LC, CH and NC correspond to pooled serum volumes at 37°C (T37). ( B ) The fluorescence intensity ratios of PHC to LC, CH and NC correspond to detection temperatures for 3 μL (S3) and 15 μL (S15) of pooled serum. ( C , D ) The fluorescence intensity ratios of PHC to LC, CH and NC correspond to EvaGreen volumes for 3 μL (S3) and 15 μL (S15) of pooled serum at 8°C (T8) and 37°C (T37). ( E , F ) The fluorescence intensity ratios of PHC to LC, CH and NC correspond to the cycle numbers (incubation time) for 3 μL of pooled serum at 8°C (S3T8) and 15 μL of pooled serum at 37°C (S15T37) in the absence of EvaGreen and for 3 μL of pooled serum at 8°C (S3T8E) and 15 μL of pooled serum at 37°C (S15T37E) in the presence of EvaGreen. PHC: primary hepatic carcinoma; LC: liver cirrhosis; CH: chronic hepatitis; NC: normal control.
Figure Legend Snippet: Optimum conditions for the fluorescence intensity measurements of pooled serum samples ( A ) The fluorescence intensity ratios of PHC to LC, CH and NC correspond to pooled serum volumes at 37°C (T37). ( B ) The fluorescence intensity ratios of PHC to LC, CH and NC correspond to detection temperatures for 3 μL (S3) and 15 μL (S15) of pooled serum. ( C , D ) The fluorescence intensity ratios of PHC to LC, CH and NC correspond to EvaGreen volumes for 3 μL (S3) and 15 μL (S15) of pooled serum at 8°C (T8) and 37°C (T37). ( E , F ) The fluorescence intensity ratios of PHC to LC, CH and NC correspond to the cycle numbers (incubation time) for 3 μL of pooled serum at 8°C (S3T8) and 15 μL of pooled serum at 37°C (S15T37) in the absence of EvaGreen and for 3 μL of pooled serum at 8°C (S3T8E) and 15 μL of pooled serum at 37°C (S15T37E) in the presence of EvaGreen. PHC: primary hepatic carcinoma; LC: liver cirrhosis; CH: chronic hepatitis; NC: normal control.

Techniques Used: Fluorescence, Incubation

13) Product Images from "Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics"

Article Title: Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics

Journal: Scientific Reports

doi: 10.1038/s41598-017-05436-4

Droplet fusion and subsequent single-cell WGA in sd-MDA. ( a ) Histograms of droplets before (Cell lysate droplet: blue) and after fusion (SAG droplet: red). ( b ) Fluorescence image of droplets after the 1 st -round MDA reaction. E. coli cells were introduced at 0.1 cells/droplet and their genomes were amplified for 2 h with Evagreen dye. Scale bar; 100 μm. (c) Time-dependent appearance of the fluorescence signal during amplification of single E. coli genome. All data are presented as averaged intensities of fluorescent positive droplets measured with SD, and 100 droplets were analyzed at each time point. ( d ) Relationship between introduced E. coli cell concentration and the number of fluorescent positive droplets.
Figure Legend Snippet: Droplet fusion and subsequent single-cell WGA in sd-MDA. ( a ) Histograms of droplets before (Cell lysate droplet: blue) and after fusion (SAG droplet: red). ( b ) Fluorescence image of droplets after the 1 st -round MDA reaction. E. coli cells were introduced at 0.1 cells/droplet and their genomes were amplified for 2 h with Evagreen dye. Scale bar; 100 μm. (c) Time-dependent appearance of the fluorescence signal during amplification of single E. coli genome. All data are presented as averaged intensities of fluorescent positive droplets measured with SD, and 100 droplets were analyzed at each time point. ( d ) Relationship between introduced E. coli cell concentration and the number of fluorescent positive droplets.

Techniques Used: Whole Genome Amplification, Multiple Displacement Amplification, Fluorescence, Amplification, Concentration Assay

14) Product Images from "Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification"

Article Title: Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification

Journal: PLoS ONE

doi: 10.1371/journal.pone.0138733

Droplet MDA of low-input lambda DNA. (a) Sequential fluorescent images of droplets encapsulating lambda DNA at a concentration of 265 ag/droplet (5 copies lambda DNA per droplet) with Evagreen dye. (b) Time-dependent appearance of the fluorescence signal during compartmentalized amplification of the denatured lambda DNA (input concentration 54 ag/droplet (1 copy lamda DNA per droplet) and 265 ag/droplet). All data are presented as averaged intensities of fluorescent positive droplets measured with SEM, and 100 droplets were analyzed at each time point.
Figure Legend Snippet: Droplet MDA of low-input lambda DNA. (a) Sequential fluorescent images of droplets encapsulating lambda DNA at a concentration of 265 ag/droplet (5 copies lambda DNA per droplet) with Evagreen dye. (b) Time-dependent appearance of the fluorescence signal during compartmentalized amplification of the denatured lambda DNA (input concentration 54 ag/droplet (1 copy lamda DNA per droplet) and 265 ag/droplet). All data are presented as averaged intensities of fluorescent positive droplets measured with SEM, and 100 droplets were analyzed at each time point.

Techniques Used: Multiple Displacement Amplification, Lambda DNA Preparation, Concentration Assay, Fluorescence, Amplification

15) Product Images from "Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR"

Article Title: Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR

Journal: Infectious Agents and Cancer

doi: 10.1186/1750-9378-5-9

Complete and minimum E6-1 preamplification mixtures used to perform E6-2 nested qPCR amplification . ( A ) Complete preamplification series. Successive stages: 1) preparation of E6-1 preamplification mixture containing; 2)
Figure Legend Snippet: Complete and minimum E6-1 preamplification mixtures used to perform E6-2 nested qPCR amplification . ( A ) Complete preamplification series. Successive stages: 1) preparation of E6-1 preamplification mixture containing; 2) "preamplification" by conventional PCR; 3) E6-2 amplification in "nested" qPCR mixtures containing EvaGreen and 1/50 volume of the E6-1 preamplified mixture. Tubes 1, a, b, c and d: positive control preamplification mixtures with serial logarithmic dilutions of pHV101 in the range of 2.5 × 10 6 -2.5 × 10 2 molecules per tube. Tube 2: Blank preamplification (without DNA). Tube 3: Problem preamplification (50 ng of SiHa DNA). Tubes 4 and 5: Negative preamplification controls (50 ng "carrier" normal human blood DNA). Asterisks indicate preamplified mixtures. ( B ) Minimum preamplification series. Successive stages: 1) preparation of E6-1 preamplification mixture including only the positive control ("calibration") with the highest pHV101 content; 2a) E6-1 "preamplification" by conventional PCR; 2b) serial logarithmic dilutions of the preamplified calibration mixture; 3) amplification of E6-2 in nested qPCR mixtures containing EvaGreen, the E6-2 primers and 1/50 volume of E6-1 preamplified mixtures. Tube 1: Positive control amplification mixture with 2.5 × 10 6 pHV101 molecules. Tube 2: Blank preamplification mixture (without DNA). Tube 3: Problem preamplification mixture (50 ng of SiHa DNA). Tubes 4 and 5: Negative preamplification controls (50 ng "carrier" normal human blood DNA). Tubes a, b, c, and d: serial logarithmic dilutions from the preamplified positive control mixture used to prepare nested qPCR mixtures equivalent to those preamplified with 2.5 × 10 5 -2.5 × 10 2 pHV101 molecules. Asterisks of numbered tubes indicate preamplified mixtures.

Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Positive Control

16) Product Images from "iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications"

Article Title: iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw083

Microfluidic-assisted screening. ( A ) Experimental workflow. Steps performed on-chip (gray boxes) were distinguished from those performed off-chip (white boxes). ( B ) PCR droplets production. Aqueous phase supplemented with a high concentration of a orange fluorescent dye was injected into droplet generator device and 2.5 pl droplets were generated by focusing aqueous (dark orange) and oil (gray) flows. Emulsions were collected and thermocycled. ( C ) Droplets fusion. Small PCR droplets were reinjected into droplet-fusion device and spaced by a stream of oil. 16 pl droplets containing In Vitro Transcription (IVT, light orange) mixture supplemented with DFHBI were concomitantly produced and synchronized with PCR droplets. Pairs of droplets were then fused when passing in between a ground-connected electrode (gnd, in black) and an electrode to which tension (pos, in red) was applied. ( D ) Droplets sorting. After incubation, emulsions were reinjected into a Fluorescence Activated Droplet Sorting device and the fluorescence of each droplet read at a detection point (blue arrow and blue line on the micrograph). Based on the fluorescence signal, droplets of interest (green) were deflected into sort channel by applying tension to one of the electrode (pos, in red) whereas non-fluorescent droplets (orange) flowed into the waste channel. ( E ) Typical fluorescence profile of screened emulsion. The analysis of DFHBI green fluorescence and Texas-Red orange fluorescence allowed identifying the different populations composing the emulsion. Indeed, using orange fluorescence signal, IVT droplets fused to single PCR droplets (populations 2–5) were easily discriminated from unfused (population 1) and double fused (population 6) IVT droplets. Green fluorescence resulting from EvaGreen intercalation allowed discriminating droplets containing amplified DNA (population 4) from droplet resulting from fusion with an initially empty PCR droplet (population 2). Finally, the stronger DFHBI green fluorescence allowed discriminating droplets containing non-fluorogenic (population 2) and highly fluorogenic aptamers (population 5, red dashed boxed). Population 5 was gated and corresponding droplets sorted. ( F ) Evolution profiles of SpiSel-derived mutants. Gene libraries obtained after mutagenesis (in red) or screening steps (in gray) were transcribed, the RNAs purified and their fluorogenic properties assayed in the presence of the salt used for the selection. Fluorescence values were normalized to that of SpiSel (black circle) in the same conditions. Screenings performed in potassium (triangles) were distinguished from those performed in sodium (squares). Values are the mean of two independent experiments and error bars correspond to ± 1 standard error.
Figure Legend Snippet: Microfluidic-assisted screening. ( A ) Experimental workflow. Steps performed on-chip (gray boxes) were distinguished from those performed off-chip (white boxes). ( B ) PCR droplets production. Aqueous phase supplemented with a high concentration of a orange fluorescent dye was injected into droplet generator device and 2.5 pl droplets were generated by focusing aqueous (dark orange) and oil (gray) flows. Emulsions were collected and thermocycled. ( C ) Droplets fusion. Small PCR droplets were reinjected into droplet-fusion device and spaced by a stream of oil. 16 pl droplets containing In Vitro Transcription (IVT, light orange) mixture supplemented with DFHBI were concomitantly produced and synchronized with PCR droplets. Pairs of droplets were then fused when passing in between a ground-connected electrode (gnd, in black) and an electrode to which tension (pos, in red) was applied. ( D ) Droplets sorting. After incubation, emulsions were reinjected into a Fluorescence Activated Droplet Sorting device and the fluorescence of each droplet read at a detection point (blue arrow and blue line on the micrograph). Based on the fluorescence signal, droplets of interest (green) were deflected into sort channel by applying tension to one of the electrode (pos, in red) whereas non-fluorescent droplets (orange) flowed into the waste channel. ( E ) Typical fluorescence profile of screened emulsion. The analysis of DFHBI green fluorescence and Texas-Red orange fluorescence allowed identifying the different populations composing the emulsion. Indeed, using orange fluorescence signal, IVT droplets fused to single PCR droplets (populations 2–5) were easily discriminated from unfused (population 1) and double fused (population 6) IVT droplets. Green fluorescence resulting from EvaGreen intercalation allowed discriminating droplets containing amplified DNA (population 4) from droplet resulting from fusion with an initially empty PCR droplet (population 2). Finally, the stronger DFHBI green fluorescence allowed discriminating droplets containing non-fluorogenic (population 2) and highly fluorogenic aptamers (population 5, red dashed boxed). Population 5 was gated and corresponding droplets sorted. ( F ) Evolution profiles of SpiSel-derived mutants. Gene libraries obtained after mutagenesis (in red) or screening steps (in gray) were transcribed, the RNAs purified and their fluorogenic properties assayed in the presence of the salt used for the selection. Fluorescence values were normalized to that of SpiSel (black circle) in the same conditions. Screenings performed in potassium (triangles) were distinguished from those performed in sodium (squares). Values are the mean of two independent experiments and error bars correspond to ± 1 standard error.

Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Concentration Assay, Injection, Generated, In Vitro, Produced, Incubation, Fluorescence, Amplification, Derivative Assay, Mutagenesis, Purification, Selection

17) Product Images from "One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV"

Article Title: One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2016.02166

EvaGreen activity for RT-LAMP. Resultant microchamber devices following the loop-mediated isothermal amplification (LAMP) reaction with EvaGreen (5 and 10X) under UV (On). DNA ladder-like LAMP amplification pattern was confirmed by 2% agarose gel electrophoresis. T: template; P: LAMP-primers. The fluorescence backgrounds were shown as red-squares.
Figure Legend Snippet: EvaGreen activity for RT-LAMP. Resultant microchamber devices following the loop-mediated isothermal amplification (LAMP) reaction with EvaGreen (5 and 10X) under UV (On). DNA ladder-like LAMP amplification pattern was confirmed by 2% agarose gel electrophoresis. T: template; P: LAMP-primers. The fluorescence backgrounds were shown as red-squares.

Techniques Used: Activity Assay, Amplification, Agarose Gel Electrophoresis, Fluorescence

Sensitivity of one-pot RT-LAMP. (A) 10X EvaGreen was initially mixed with RT-LAMP reagents, and the reaction was performed and visualized in microchamber. The purified MERS-CoV RNA was serially diluted in the range 4 × 10 3 –4 × 10 -1 /μL. Relative fluorescent signals were analyzed by using ImageJ software. The relative intensity of fluorescence for positive amplification was normalized to the controls (-Template/-Primer). Red square indicates background fluorescence. (B) Agarose gel electrophoresis confirmation indicated that one-pot RT-LAMP can detect MERS-CoV as few as 0.4 RNA copies. DNA ladder-like pattern was confirmed by 2% agarose gel electrophoresis. T, template; P, primer.
Figure Legend Snippet: Sensitivity of one-pot RT-LAMP. (A) 10X EvaGreen was initially mixed with RT-LAMP reagents, and the reaction was performed and visualized in microchamber. The purified MERS-CoV RNA was serially diluted in the range 4 × 10 3 –4 × 10 -1 /μL. Relative fluorescent signals were analyzed by using ImageJ software. The relative intensity of fluorescence for positive amplification was normalized to the controls (-Template/-Primer). Red square indicates background fluorescence. (B) Agarose gel electrophoresis confirmation indicated that one-pot RT-LAMP can detect MERS-CoV as few as 0.4 RNA copies. DNA ladder-like pattern was confirmed by 2% agarose gel electrophoresis. T, template; P, primer.

Techniques Used: Purification, Software, Fluorescence, Amplification, Agarose Gel Electrophoresis

Specificity of one-pot RT-LAMP for MERS-CoV. RNA samples of MERS-CoV and the other respiratory pathogens were used for specificity evaluation of one-pot RT-LAMP system for MERS-CoV detection; fluorescence was visualized by 10X EvaGreen in microchamber. Each set of lane indicates a specific virus: lane 1, MERS-CoV Korean isolate; lane 2, Human coronavirus (HCoV)-229E; lane 3, Human metapneumovirus (HMPV); lane 4, B/Brisbane/60/2008 (Victoria lineage); lane 5, B/Phuket/3073/2013 (Yamagata lineage); lane 6, Influenza A virus (A/California/04/2009, H1N1); lane 7, influenza A virus (A/Perth/16/2009, H3N2). M, 100 bp DNA ladder.
Figure Legend Snippet: Specificity of one-pot RT-LAMP for MERS-CoV. RNA samples of MERS-CoV and the other respiratory pathogens were used for specificity evaluation of one-pot RT-LAMP system for MERS-CoV detection; fluorescence was visualized by 10X EvaGreen in microchamber. Each set of lane indicates a specific virus: lane 1, MERS-CoV Korean isolate; lane 2, Human coronavirus (HCoV)-229E; lane 3, Human metapneumovirus (HMPV); lane 4, B/Brisbane/60/2008 (Victoria lineage); lane 5, B/Phuket/3073/2013 (Yamagata lineage); lane 6, Influenza A virus (A/California/04/2009, H1N1); lane 7, influenza A virus (A/Perth/16/2009, H3N2). M, 100 bp DNA ladder.

Techniques Used: Fluorescence

18) Product Images from "Quantification of Mitochondrial DNA (mtDNA) Damage and Error Rates by Real-time QPCR"

Article Title: Quantification of Mitochondrial DNA (mtDNA) Damage and Error Rates by Real-time QPCR

Journal: Mitochondrion

doi: 10.1016/j.mito.2008.11.004

LRCPR of mtDNA using increasing concentrations of EvaGreen. A. Crossing point analysis of the LRPCR using increasing concentrations of EvaGreen. 100ng of total DNA was used as described in Methods. B. 10% of the LRPCR product after 40 cycles of amplification was electrophoresed on a 1% agarose gel. The integrated density value (IDV) of the bands was determined using AlphaInnotech/AlphaEaseFC software following 40 cycles of amplification.
Figure Legend Snippet: LRCPR of mtDNA using increasing concentrations of EvaGreen. A. Crossing point analysis of the LRPCR using increasing concentrations of EvaGreen. 100ng of total DNA was used as described in Methods. B. 10% of the LRPCR product after 40 cycles of amplification was electrophoresed on a 1% agarose gel. The integrated density value (IDV) of the bands was determined using AlphaInnotech/AlphaEaseFC software following 40 cycles of amplification.

Techniques Used: Amplification, Agarose Gel Electrophoresis, Software

Comparison of realtime LRPCR and step cycle amplification of mtDNA. A. Representative picture of step LRPCR; 100 ng of total DNA was amplified using the LRPCR protocol and the reaction tube was removed at the cycle number indicated above each band. 10% of the LRPCR product was run on a 1% agarose gel. B. Realtime determination of DNA fluorescence (n=4) was determined as described in Methods. For the step cycle, the integrated density value (IDV) of the bands (n=3 for each step) was determined using AlphaInnotech/AlphaEaseFC software. All individual reactions contained 1:1250 dilution of EvaGreen. Values represented are mean±SEM of fluorescence normalized to the plateau values.
Figure Legend Snippet: Comparison of realtime LRPCR and step cycle amplification of mtDNA. A. Representative picture of step LRPCR; 100 ng of total DNA was amplified using the LRPCR protocol and the reaction tube was removed at the cycle number indicated above each band. 10% of the LRPCR product was run on a 1% agarose gel. B. Realtime determination of DNA fluorescence (n=4) was determined as described in Methods. For the step cycle, the integrated density value (IDV) of the bands (n=3 for each step) was determined using AlphaInnotech/AlphaEaseFC software. All individual reactions contained 1:1250 dilution of EvaGreen. Values represented are mean±SEM of fluorescence normalized to the plateau values.

Techniques Used: Amplification, Agarose Gel Electrophoresis, Fluorescence, Software

19) Product Images from "SYTO dyes and EvaGreen outperform SYBR Green in real-time PCR"

Article Title: SYTO dyes and EvaGreen outperform SYBR Green in real-time PCR

Journal: BMC Research Notes

doi: 10.1186/1756-0500-4-263

Melt curves for SYBR Green, EvaGreen, and SYTO 16 . All reactions contained 1000 pg shrimp genomic DNA amplified using the 16S primer set. Similar data were obtained for all experiments and amounts of template DNA tested.
Figure Legend Snippet: Melt curves for SYBR Green, EvaGreen, and SYTO 16 . All reactions contained 1000 pg shrimp genomic DNA amplified using the 16S primer set. Similar data were obtained for all experiments and amounts of template DNA tested.

Techniques Used: SYBR Green Assay, Amplification

20) Product Images from "Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR"

Article Title: Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR

Journal: Analytical and Bioanalytical Chemistry

doi: 10.1007/s00216-018-0931-z

EvaGreen real-time polymerase chain reaction results with different amounts of whole blood in the reactions. Two different assays were applied, targeting either a the invA gene of Salmonella enterica serovar Typhimurium DNA with 0.052 ng DNA added or b the RB1 gene of human DNA with 2 ng DNA added
Figure Legend Snippet: EvaGreen real-time polymerase chain reaction results with different amounts of whole blood in the reactions. Two different assays were applied, targeting either a the invA gene of Salmonella enterica serovar Typhimurium DNA with 0.052 ng DNA added or b the RB1 gene of human DNA with 2 ng DNA added

Techniques Used: Real-time Polymerase Chain Reaction

EvaGreen real-time polymerase chain reaction results with different amounts of immunoglobulin G (IgG). The assay targeting the invA gene of Salmonella enterica serovar Typhimurium was applied, and 52 pg DNA was added to the reactions. a Real-time polymerase chain reaction amplification curves with increasing amounts of IgG and b the generated Cq values, with error bars representing the standard deviation, n = 3. Cq quantification cycle
Figure Legend Snippet: EvaGreen real-time polymerase chain reaction results with different amounts of immunoglobulin G (IgG). The assay targeting the invA gene of Salmonella enterica serovar Typhimurium was applied, and 52 pg DNA was added to the reactions. a Real-time polymerase chain reaction amplification curves with increasing amounts of IgG and b the generated Cq values, with error bars representing the standard deviation, n = 3. Cq quantification cycle

Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Generated, Standard Deviation

21) Product Images from "Inhibitory Effect of Bridged Nucleosides on Thermus aquaticus DNA Polymerase and Insight into the Binding Interactions"

Article Title: Inhibitory Effect of Bridged Nucleosides on Thermus aquaticus DNA Polymerase and Insight into the Binding Interactions

Journal: PLoS ONE

doi: 10.1371/journal.pone.0147234

Determination of IC 50 values. (A) IC 50 value of Taq DNA polymerase by 2',4'-bridged thymidine using a primer extension assay with EvaGreen. The concentrations of 2',4'-bridged thymidine were used at 0.01, 10, 50, and 500 μM (B) IC 50 value of Taq DNA polymerase by ddCTP using a primer extension assay with EvaGreen. The concentrations of 2',4'-bridged thymidine were used at 0.1, 50, 250, and 2500 μM (C) IC 50 value of Taq DNA polymerase by 2',4'-bridged thymidine using the TaqMan method. The concentrations of 2',4'-bridged thymidine were used at 0.01, 1, 10, and 500 μM
Figure Legend Snippet: Determination of IC 50 values. (A) IC 50 value of Taq DNA polymerase by 2',4'-bridged thymidine using a primer extension assay with EvaGreen. The concentrations of 2',4'-bridged thymidine were used at 0.01, 10, 50, and 500 μM (B) IC 50 value of Taq DNA polymerase by ddCTP using a primer extension assay with EvaGreen. The concentrations of 2',4'-bridged thymidine were used at 0.1, 50, 250, and 2500 μM (C) IC 50 value of Taq DNA polymerase by 2',4'-bridged thymidine using the TaqMan method. The concentrations of 2',4'-bridged thymidine were used at 0.01, 1, 10, and 500 μM

Techniques Used: Primer Extension Assay

22) Product Images from "Melting Temperature Mapping Method: A Novel Method for Rapid Identification of Unknown Pathogenic Microorganisms within Three Hours of Sample Collection"

Article Title: Melting Temperature Mapping Method: A Novel Method for Rapid Identification of Unknown Pathogenic Microorganisms within Three Hours of Sample Collection

Journal: Scientific Reports

doi: 10.1038/srep12543

Concept of the Tm mapping method. ( A ) The strategy for the primer designs is shown. Nested PCR is performed using seven bacterial universal primer sets, and then the seven Tm values are obtained. ( B ) Mapping the seven Tm values on two dimensions leads to the identification of the unique bacterial species-specific shape. The average of all seven Tm values includes the measurement error among trials; however, the Tm mapping shape is not affected by this type of error. ( C ) Using an analytical instrument with a high degree of thermal accuracy among PCR tubes and Tm value analysis with EvaGreen dye in 36 samples of the same bacterial DNA in the same trial, the tube-to-tube variation is within ±0.1 °C. ( D ) In order to analyze the Tm mapping “shape”, we developed a method to measure the distance of each individual Tm value from the average value. Tm values above the average receive a “+” designation, while those below the average receive a “−” designation. The Tm mapping shape is identified by comparing the seven distances obtained from the unknown bacteria to those in the database. ( E ) In order to identify a bacterial isolate, the identification software program calculates the Difference Values using the indicated formula. The closer the Difference Value is to zero, the more similar the Tm mapping shape is to the shape of a given species of pathogenic bacteria in the database.
Figure Legend Snippet: Concept of the Tm mapping method. ( A ) The strategy for the primer designs is shown. Nested PCR is performed using seven bacterial universal primer sets, and then the seven Tm values are obtained. ( B ) Mapping the seven Tm values on two dimensions leads to the identification of the unique bacterial species-specific shape. The average of all seven Tm values includes the measurement error among trials; however, the Tm mapping shape is not affected by this type of error. ( C ) Using an analytical instrument with a high degree of thermal accuracy among PCR tubes and Tm value analysis with EvaGreen dye in 36 samples of the same bacterial DNA in the same trial, the tube-to-tube variation is within ±0.1 °C. ( D ) In order to analyze the Tm mapping “shape”, we developed a method to measure the distance of each individual Tm value from the average value. Tm values above the average receive a “+” designation, while those below the average receive a “−” designation. The Tm mapping shape is identified by comparing the seven distances obtained from the unknown bacteria to those in the database. ( E ) In order to identify a bacterial isolate, the identification software program calculates the Difference Values using the indicated formula. The closer the Difference Value is to zero, the more similar the Tm mapping shape is to the shape of a given species of pathogenic bacteria in the database.

Techniques Used: Nested PCR, Polymerase Chain Reaction, Software

23) Product Images from "Programming an in vitro DNA oscillator using a molecular networking strategy"

Article Title: Programming an in vitro DNA oscillator using a molecular networking strategy

Journal: Molecular Systems Biology

doi: 10.1038/msb.2010.120

Experimental assembly. All the reactions shown were performed at 38.5°C, initiated with 0.1 nM α and monitored (ex. 490 nM; em. 510 nM) using EvaGreen-induced fluorescence. ( A ) One-node positive-feedback loop (autocatalytic module). In the presence of Bst Polymerase (80 U ml −1 ) and nicking enzyme Nt.bstNBI (200 U ml −1 ), template T 1 (60 nM) performs an exponential amplification of its input α. The fluorescence reaches a plateau when the template gets saturated with α. The low subsequent increase is due to the accumulation of single-stranded α, weakly fluorescent in these conditions. In the presence of exonuclease RecJ f (30 U ml −1 ), the reaction reaches a flat steady state instead. ( B ) Inhibited amplification. Increasing amounts of inhibitor (from 0 to 1 eq. of T 1 ) decrease the amplification rate of the previous system (−RecJ f ). ( C ) Oscillator. Production of Inh is connected to the presence of α as in Figure 1F . This three-templates (T 1 and T 3 : 30 nM; T 2 : 5 nM) three-enzymes (Bst, Nt.BstNBI, RecJ f ) system produces sustained fluorescent oscillations with a period of 100 min, in good agreement with the predicted evolution of the total concentration of base pairs ( D ).
Figure Legend Snippet: Experimental assembly. All the reactions shown were performed at 38.5°C, initiated with 0.1 nM α and monitored (ex. 490 nM; em. 510 nM) using EvaGreen-induced fluorescence. ( A ) One-node positive-feedback loop (autocatalytic module). In the presence of Bst Polymerase (80 U ml −1 ) and nicking enzyme Nt.bstNBI (200 U ml −1 ), template T 1 (60 nM) performs an exponential amplification of its input α. The fluorescence reaches a plateau when the template gets saturated with α. The low subsequent increase is due to the accumulation of single-stranded α, weakly fluorescent in these conditions. In the presence of exonuclease RecJ f (30 U ml −1 ), the reaction reaches a flat steady state instead. ( B ) Inhibited amplification. Increasing amounts of inhibitor (from 0 to 1 eq. of T 1 ) decrease the amplification rate of the previous system (−RecJ f ). ( C ) Oscillator. Production of Inh is connected to the presence of α as in Figure 1F . This three-templates (T 1 and T 3 : 30 nM; T 2 : 5 nM) three-enzymes (Bst, Nt.BstNBI, RecJ f ) system produces sustained fluorescent oscillations with a period of 100 min, in good agreement with the predicted evolution of the total concentration of base pairs ( D ).

Techniques Used: Fluorescence, Amplification, Concentration Assay

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Amplification:

Article Title: A rapid, low-cost, and microfluidic chip-based system for parallel identification of multiple pathogens related to clinical pneumonia
Article Snippet: .. Isothermal amplification assays Each 10-μL isothermal nucleic acid amplification assay for pathogen molecular diagnostics consisted of 0.2 μM each of F3 and B3, 1.6 μM each of FIP and BIP, 0.4 μM each of LF and LB, 8 U of Bst DNA Polymerase (Large Fragment), 0.1 mM dUTP, 0.4 mM dNTPs (New England Biolabs Ltd., Beverly, USA), 0.5 mg/ml BSA (Fluka Sigma-Aldrich Inc., Missouri, USA), 0.6× EvaGreen (Biotium Inc., California, USA), 0.8 M betaine (Fluka Sigma-Aldrich Inc., Missouri, USA), 6 mM MgSO4 (Beijing Chemical Reagents Company, Beijing, China), 0.1 U/mL Uracil-DNA Glycosylase (Fermentas Inc., Burlington, Canada), 10 mM (NH4 )2 SO4 , 20 mM Tris-HCl (pH 8.8 at 25 °C), 10 mM KCl, 0.1% Triton X-100, and 2 μL template DNA. ..

Article Title: Efficiency of whole genome amplification of single circulating tumor cells enriched by CellSearch and sorted by FACS
Article Snippet: .. After this step, 5 μl of an amplification mix containing Evagreen, a double-stranded DNA dye (Biotium, Hayward, CA, USA; cat. 31000), was added to each well. .. The amplification mix consisted of components from the GE Illustra GenomiPhi DNA amplification kit (GE Healthcare Life Sciences, Waukesha, WI, USA; cat. 25-6600-31) combined with Evagreen to monitor the reaction.

Polymerase Chain Reaction:

Article Title: A high-throughput qPCR system for simultaneous quantitative detection of dairy Lactococcus lactis and Leuconostoc bacteriophages
Article Snippet: .. PCR reactions for comparison of SYBR Green I (Thermo Fisher Scientific) and EvaGreen (Biotium, USA) detection chemistries were prepared as described above using the P220 phage DNA as template. ..

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    Biotium evagreen
    Reverse transcriptase assays. A. Fluorogenic assay. RT activity was measured by detection of RNA:DNA heteroduplex by fluorescence of <t>EvaGreen</t> binding. Oligo dT primed poly A was incubated at 37°C and 65°C in the presence of indicated Pol enzymes in manufacturer recommended buffers and dTTP. Fluorescence measurements were obtained every 6 seconds for 10 minutes. The initial slopes from a plot of RFU vs. time in seconds were determined by linear least square regression from 30 to 150 seconds at 37°C and from 30 to 90 seconds at 65°C. Error bars are standard error of regression slope. B. RT primer extension assay. HEX-labeled dT 20 primed poly A was incubated 10 minutes at 37°C and then 10 minutes at 65°C in the presence of indicated Pol enzymes and dTTP in manufacturer recommended buffers. Primer extension products were resolved by 10% denaturing PAGE and imaged on a Molecular Imager FX (Bio-Rad). Left facing triangle indicates migration of unextended dT20 primer and asterisk indicates bromophenol blue dye front. C. RT MS2-specific primer extension. 5′-labeled primer was annealed to MS2 RNA and incubated 10 minutes at 37°C and then 30 minutes at 65°C in the presence of indicated Pol enzymes with dNTPS (N = A,C,G,T) in manufacturer recommended buffers. Primer extension products were resolved by 5% denaturing PAGE. Lane 1 No RNA+MMLV RT; Lane 2: MS2 RNA No RT; Lane 3 MS2 RNA+MMLV RT, Lane 3 MS2 RNA+3173 Pol. Molecular weight in bases indicted. Red Arrow: ∼650 base MMLV extension product. Blue Arrow: ∼715 base PyroScript extension product. Green arrow: Non-templated MMLV reaction product.
    Evagreen, supplied by Biotium, used in various techniques. Bioz Stars score: 94/100, based on 124 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Biotium dye evagreen
    Effect of dNTPs on T m measurement of DNA duplexes by HRM. The buffer contains 1x <t>EvaGreen,</t> 10 mM phosphate buffer (pH 7.4), and 100 mM NaCl. In this assay, 0, 0.02, 0.2, or 2 mM dNTPs were used.
    Dye Evagreen, supplied by Biotium, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dye evagreen/product/Biotium
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Reverse transcriptase assays. A. Fluorogenic assay. RT activity was measured by detection of RNA:DNA heteroduplex by fluorescence of EvaGreen binding. Oligo dT primed poly A was incubated at 37°C and 65°C in the presence of indicated Pol enzymes in manufacturer recommended buffers and dTTP. Fluorescence measurements were obtained every 6 seconds for 10 minutes. The initial slopes from a plot of RFU vs. time in seconds were determined by linear least square regression from 30 to 150 seconds at 37°C and from 30 to 90 seconds at 65°C. Error bars are standard error of regression slope. B. RT primer extension assay. HEX-labeled dT 20 primed poly A was incubated 10 minutes at 37°C and then 10 minutes at 65°C in the presence of indicated Pol enzymes and dTTP in manufacturer recommended buffers. Primer extension products were resolved by 10% denaturing PAGE and imaged on a Molecular Imager FX (Bio-Rad). Left facing triangle indicates migration of unextended dT20 primer and asterisk indicates bromophenol blue dye front. C. RT MS2-specific primer extension. 5′-labeled primer was annealed to MS2 RNA and incubated 10 minutes at 37°C and then 30 minutes at 65°C in the presence of indicated Pol enzymes with dNTPS (N = A,C,G,T) in manufacturer recommended buffers. Primer extension products were resolved by 5% denaturing PAGE. Lane 1 No RNA+MMLV RT; Lane 2: MS2 RNA No RT; Lane 3 MS2 RNA+MMLV RT, Lane 3 MS2 RNA+3173 Pol. Molecular weight in bases indicted. Red Arrow: ∼650 base MMLV extension product. Blue Arrow: ∼715 base PyroScript extension product. Green arrow: Non-templated MMLV reaction product.

    Journal: PLoS ONE

    Article Title: Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme

    doi: 10.1371/journal.pone.0038371

    Figure Lengend Snippet: Reverse transcriptase assays. A. Fluorogenic assay. RT activity was measured by detection of RNA:DNA heteroduplex by fluorescence of EvaGreen binding. Oligo dT primed poly A was incubated at 37°C and 65°C in the presence of indicated Pol enzymes in manufacturer recommended buffers and dTTP. Fluorescence measurements were obtained every 6 seconds for 10 minutes. The initial slopes from a plot of RFU vs. time in seconds were determined by linear least square regression from 30 to 150 seconds at 37°C and from 30 to 90 seconds at 65°C. Error bars are standard error of regression slope. B. RT primer extension assay. HEX-labeled dT 20 primed poly A was incubated 10 minutes at 37°C and then 10 minutes at 65°C in the presence of indicated Pol enzymes and dTTP in manufacturer recommended buffers. Primer extension products were resolved by 10% denaturing PAGE and imaged on a Molecular Imager FX (Bio-Rad). Left facing triangle indicates migration of unextended dT20 primer and asterisk indicates bromophenol blue dye front. C. RT MS2-specific primer extension. 5′-labeled primer was annealed to MS2 RNA and incubated 10 minutes at 37°C and then 30 minutes at 65°C in the presence of indicated Pol enzymes with dNTPS (N = A,C,G,T) in manufacturer recommended buffers. Primer extension products were resolved by 5% denaturing PAGE. Lane 1 No RNA+MMLV RT; Lane 2: MS2 RNA No RT; Lane 3 MS2 RNA+MMLV RT, Lane 3 MS2 RNA+3173 Pol. Molecular weight in bases indicted. Red Arrow: ∼650 base MMLV extension product. Blue Arrow: ∼715 base PyroScript extension product. Green arrow: Non-templated MMLV reaction product.

    Article Snippet: For the Roche and the Lucigen reagents, a fluorescent DNA-binding dye, EvaGreen (Biotium, Hayward, CA), was added at 0.5×.

    Techniques: Activity Assay, Fluorescence, Binding Assay, Incubation, Primer Extension Assay, Labeling, Polyacrylamide Gel Electrophoresis, Migration, Molecular Weight

    Structure of the microfluidic chip and method for the parallel identification of multiple pathogens. ( A ) Basement of the microfluidic chip. ( B ) Cover of the microfluidic chip. ( C ) Six primers embedded together at the bottom of one test cell using low melting point Sepharose CL-4B. ( D ) The mixture of the prepared DNA sample and isothermal nucleic acid amplification reactants is injected into the microfluidic chip via the inlet hole using a pipette. ( E ) The mixtures after being centrifuged at 5000 rpm. ( F ) Six primers released at > 50 °C. ( G ) The fluorescent marker EvaGreen bound to the amplified products as nucleic acid amplification occurred at 65 °C.

    Journal: Scientific Reports

    Article Title: A rapid, low-cost, and microfluidic chip-based system for parallel identification of multiple pathogens related to clinical pneumonia

    doi: 10.1038/s41598-017-06739-2

    Figure Lengend Snippet: Structure of the microfluidic chip and method for the parallel identification of multiple pathogens. ( A ) Basement of the microfluidic chip. ( B ) Cover of the microfluidic chip. ( C ) Six primers embedded together at the bottom of one test cell using low melting point Sepharose CL-4B. ( D ) The mixture of the prepared DNA sample and isothermal nucleic acid amplification reactants is injected into the microfluidic chip via the inlet hole using a pipette. ( E ) The mixtures after being centrifuged at 5000 rpm. ( F ) Six primers released at > 50 °C. ( G ) The fluorescent marker EvaGreen bound to the amplified products as nucleic acid amplification occurred at 65 °C.

    Article Snippet: Isothermal amplification assays Each 10-μL isothermal nucleic acid amplification assay for pathogen molecular diagnostics consisted of 0.2 μM each of F3 and B3, 1.6 μM each of FIP and BIP, 0.4 μM each of LF and LB, 8 U of Bst DNA Polymerase (Large Fragment), 0.1 mM dUTP, 0.4 mM dNTPs (New England Biolabs Ltd., Beverly, USA), 0.5 mg/ml BSA (Fluka Sigma-Aldrich Inc., Missouri, USA), 0.6× EvaGreen (Biotium Inc., California, USA), 0.8 M betaine (Fluka Sigma-Aldrich Inc., Missouri, USA), 6 mM MgSO4 (Beijing Chemical Reagents Company, Beijing, China), 0.1 U/mL Uracil-DNA Glycosylase (Fermentas Inc., Burlington, Canada), 10 mM (NH4 )2 SO4 , 20 mM Tris-HCl (pH 8.8 at 25 °C), 10 mM KCl, 0.1% Triton X-100, and 2 μL template DNA.

    Techniques: Chromatin Immunoprecipitation, Amplification, Injection, Transferring, Marker

    Performance of SYBR Green I and EvaGreen detection chemistries during qPCR assays. (A) Standard curves generated from amplification of serially diluted L . lactis phage P220 genome detected with the corresponding chemistries; (B) the corresponding performance parameters; and (C) amplification plots detected with SYBR Green I (brown) and EvaGreen (green).

    Journal: PLoS ONE

    Article Title: A high-throughput qPCR system for simultaneous quantitative detection of dairy Lactococcus lactis and Leuconostoc bacteriophages

    doi: 10.1371/journal.pone.0174223

    Figure Lengend Snippet: Performance of SYBR Green I and EvaGreen detection chemistries during qPCR assays. (A) Standard curves generated from amplification of serially diluted L . lactis phage P220 genome detected with the corresponding chemistries; (B) the corresponding performance parameters; and (C) amplification plots detected with SYBR Green I (brown) and EvaGreen (green).

    Article Snippet: PCR reactions for comparison of SYBR Green I (Thermo Fisher Scientific) and EvaGreen (Biotium, USA) detection chemistries were prepared as described above using the P220 phage DNA as template.

    Techniques: SYBR Green Assay, Real-time Polymerase Chain Reaction, Generated, Amplification

    Effect of dNTPs on T m measurement of DNA duplexes by HRM. The buffer contains 1x EvaGreen, 10 mM phosphate buffer (pH 7.4), and 100 mM NaCl. In this assay, 0, 0.02, 0.2, or 2 mM dNTPs were used.

    Journal: Journal of Analytical Methods in Chemistry

    Article Title: Assessment for Melting Temperature Measurement of Nucleic Acid by HRM

    doi: 10.1155/2016/5318935

    Figure Lengend Snippet: Effect of dNTPs on T m measurement of DNA duplexes by HRM. The buffer contains 1x EvaGreen, 10 mM phosphate buffer (pH 7.4), and 100 mM NaCl. In this assay, 0, 0.02, 0.2, or 2 mM dNTPs were used.

    Article Snippet: Materials and Reagents DNA and RNA oligonucleotides (in Tables and ) were synthesized from Invitrogen (Shanghai, China); the fluorescent dye EvaGreen 20x was obtained from Biotium (Hayward, CA).

    Techniques:

    Effect of EvaGreen concentration on T m of DNA duplex by HRM. DNA duplexes GC3/15 (a) or GC12/15 (b) of 1 μ M were measured in the solution with 0.25x, 0.5x, 1x, 2x, or 5x EvaGreen, 10 mM phosphate (pH 7.4), and 100 mM NaCl. Comparisons were done using one-way ANOVA analysis; ∗ P

    Journal: Journal of Analytical Methods in Chemistry

    Article Title: Assessment for Melting Temperature Measurement of Nucleic Acid by HRM

    doi: 10.1155/2016/5318935

    Figure Lengend Snippet: Effect of EvaGreen concentration on T m of DNA duplex by HRM. DNA duplexes GC3/15 (a) or GC12/15 (b) of 1 μ M were measured in the solution with 0.25x, 0.5x, 1x, 2x, or 5x EvaGreen, 10 mM phosphate (pH 7.4), and 100 mM NaCl. Comparisons were done using one-way ANOVA analysis; ∗ P

    Article Snippet: Materials and Reagents DNA and RNA oligonucleotides (in Tables and ) were synthesized from Invitrogen (Shanghai, China); the fluorescent dye EvaGreen 20x was obtained from Biotium (Hayward, CA).

    Techniques: Concentration Assay

    Fluorescence melting curves (a) and differential curves (b) of 6–20 bp DNA duplexes. Each DNA duplex (1 μ M) was measured in a 10 μ L solution containing 1x EvaGreen, 10 mM phosphate (pH 7.4), and 100 mM NaCl.

    Journal: Journal of Analytical Methods in Chemistry

    Article Title: Assessment for Melting Temperature Measurement of Nucleic Acid by HRM

    doi: 10.1155/2016/5318935

    Figure Lengend Snippet: Fluorescence melting curves (a) and differential curves (b) of 6–20 bp DNA duplexes. Each DNA duplex (1 μ M) was measured in a 10 μ L solution containing 1x EvaGreen, 10 mM phosphate (pH 7.4), and 100 mM NaCl.

    Article Snippet: Materials and Reagents DNA and RNA oligonucleotides (in Tables and ) were synthesized from Invitrogen (Shanghai, China); the fluorescent dye EvaGreen 20x was obtained from Biotium (Hayward, CA).

    Techniques: Fluorescence