dntp mix  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier
Bioz Manufacturer Symbol Thermo Fisher manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99

    Structured Review

    Thermo Fisher dntp mix
    Visualization of PSR products using (A) . SYBR Green I, (B) . GoodView, and (C) . HNB. PSR was done using crude lysate of T. palmi as positive (+ve) and water as negative (-ve) control. Addition of 1 µl of SYBR Green I and GoodView after completion of PSR reaction showed fluorescence under UV light in positive samples, whereas no fluorescence was observed in negative samples. The PSR reaction was <t>mixed</t> with 2 µl of 3 mM HNB prior to amplification and showed a change in color from violet to sky blue in the case of T. palmi , while no corresponding color change was observed in negative samples.
    Dntp Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dntp mix/product/Thermo Fisher
    Average 99 stars, based on 40 article reviews
    Price from $9.99 to $1999.99
    dntp mix - by Bioz Stars, 2022-08
    99/100 stars

    Images

    1) Product Images from "Development of a Polymerase Spiral Reaction-Based Isothermal Assay for Rapid Identification of Thrips palmi"

    Article Title: Development of a Polymerase Spiral Reaction-Based Isothermal Assay for Rapid Identification of Thrips palmi

    Journal: Frontiers in Molecular Biosciences

    doi: 10.3389/fmolb.2022.853339

    Visualization of PSR products using (A) . SYBR Green I, (B) . GoodView, and (C) . HNB. PSR was done using crude lysate of T. palmi as positive (+ve) and water as negative (-ve) control. Addition of 1 µl of SYBR Green I and GoodView after completion of PSR reaction showed fluorescence under UV light in positive samples, whereas no fluorescence was observed in negative samples. The PSR reaction was mixed with 2 µl of 3 mM HNB prior to amplification and showed a change in color from violet to sky blue in the case of T. palmi , while no corresponding color change was observed in negative samples.
    Figure Legend Snippet: Visualization of PSR products using (A) . SYBR Green I, (B) . GoodView, and (C) . HNB. PSR was done using crude lysate of T. palmi as positive (+ve) and water as negative (-ve) control. Addition of 1 µl of SYBR Green I and GoodView after completion of PSR reaction showed fluorescence under UV light in positive samples, whereas no fluorescence was observed in negative samples. The PSR reaction was mixed with 2 µl of 3 mM HNB prior to amplification and showed a change in color from violet to sky blue in the case of T. palmi , while no corresponding color change was observed in negative samples.

    Techniques Used: SYBR Green Assay, Fluorescence, Amplification

    Workflow of PSR assay for rapid, on-site detection of T. palmi . (A) . Single adult T. palmi collected from a leaf using a Camel hairbrush and placed in a 1.5 mlmicrocentrifuge tube, (B) . Thrips crushed with the help of a micro-pestle in sterile distilled water, (C) . Micro-centrifuge tube placed in a floating rack and incubated at 100°C in a water bath for 2 min, (D) . PSR mixture prepared in 0.5 ml PCR tubes, (E) . PSR reaction incubated in a water bath at 65°C for 60 min. Visual detection of presence or absence of T. palmi using (F) . SYBR Green I, (G) . GoodView. Fluorescence was observed in the positive samples while no fluorescence was detected in negative samples, (H) . Visual detection of T. palmi PSR products using HNB. Change in color from violet to sky blue indicates the presence of T. palmi , while no color change was observed in negative samples.
    Figure Legend Snippet: Workflow of PSR assay for rapid, on-site detection of T. palmi . (A) . Single adult T. palmi collected from a leaf using a Camel hairbrush and placed in a 1.5 mlmicrocentrifuge tube, (B) . Thrips crushed with the help of a micro-pestle in sterile distilled water, (C) . Micro-centrifuge tube placed in a floating rack and incubated at 100°C in a water bath for 2 min, (D) . PSR mixture prepared in 0.5 ml PCR tubes, (E) . PSR reaction incubated in a water bath at 65°C for 60 min. Visual detection of presence or absence of T. palmi using (F) . SYBR Green I, (G) . GoodView. Fluorescence was observed in the positive samples while no fluorescence was detected in negative samples, (H) . Visual detection of T. palmi PSR products using HNB. Change in color from violet to sky blue indicates the presence of T. palmi , while no color change was observed in negative samples.

    Techniques Used: Incubation, Polymerase Chain Reaction, SYBR Green Assay, Fluorescence

    2) Product Images from "Development of a Polymerase Spiral Reaction-Based Isothermal Assay for Rapid Identification of Thrips palmi"

    Article Title: Development of a Polymerase Spiral Reaction-Based Isothermal Assay for Rapid Identification of Thrips palmi

    Journal: Frontiers in Molecular Biosciences

    doi: 10.3389/fmolb.2022.853339

    Visualization of PSR products using (A) . SYBR Green I, (B) . GoodView, and (C) . HNB. PSR was done using crude lysate of T. palmi as positive (+ve) and water as negative (-ve) control. Addition of 1 µl of SYBR Green I and GoodView after completion of PSR reaction showed fluorescence under UV light in positive samples, whereas no fluorescence was observed in negative samples. The PSR reaction was mixed with 2 µl of 3 mM HNB prior to amplification and showed a change in color from violet to sky blue in the case of T. palmi , while no corresponding color change was observed in negative samples.
    Figure Legend Snippet: Visualization of PSR products using (A) . SYBR Green I, (B) . GoodView, and (C) . HNB. PSR was done using crude lysate of T. palmi as positive (+ve) and water as negative (-ve) control. Addition of 1 µl of SYBR Green I and GoodView after completion of PSR reaction showed fluorescence under UV light in positive samples, whereas no fluorescence was observed in negative samples. The PSR reaction was mixed with 2 µl of 3 mM HNB prior to amplification and showed a change in color from violet to sky blue in the case of T. palmi , while no corresponding color change was observed in negative samples.

    Techniques Used: SYBR Green Assay, Fluorescence, Amplification

    Workflow of PSR assay for rapid, on-site detection of T. palmi . (A) . Single adult T. palmi collected from a leaf using a Camel hairbrush and placed in a 1.5 mlmicrocentrifuge tube, (B) . Thrips crushed with the help of a micro-pestle in sterile distilled water, (C) . Micro-centrifuge tube placed in a floating rack and incubated at 100°C in a water bath for 2 min, (D) . PSR mixture prepared in 0.5 ml PCR tubes, (E) . PSR reaction incubated in a water bath at 65°C for 60 min. Visual detection of presence or absence of T. palmi using (F) . SYBR Green I, (G) . GoodView. Fluorescence was observed in the positive samples while no fluorescence was detected in negative samples, (H) . Visual detection of T. palmi PSR products using HNB. Change in color from violet to sky blue indicates the presence of T. palmi , while no color change was observed in negative samples.
    Figure Legend Snippet: Workflow of PSR assay for rapid, on-site detection of T. palmi . (A) . Single adult T. palmi collected from a leaf using a Camel hairbrush and placed in a 1.5 mlmicrocentrifuge tube, (B) . Thrips crushed with the help of a micro-pestle in sterile distilled water, (C) . Micro-centrifuge tube placed in a floating rack and incubated at 100°C in a water bath for 2 min, (D) . PSR mixture prepared in 0.5 ml PCR tubes, (E) . PSR reaction incubated in a water bath at 65°C for 60 min. Visual detection of presence or absence of T. palmi using (F) . SYBR Green I, (G) . GoodView. Fluorescence was observed in the positive samples while no fluorescence was detected in negative samples, (H) . Visual detection of T. palmi PSR products using HNB. Change in color from violet to sky blue indicates the presence of T. palmi , while no color change was observed in negative samples.

    Techniques Used: Incubation, Polymerase Chain Reaction, SYBR Green Assay, Fluorescence

    3) Product Images from "Development of a Polymerase Spiral Reaction-Based Isothermal Assay for Rapid Identification of Thrips palmi"

    Article Title: Development of a Polymerase Spiral Reaction-Based Isothermal Assay for Rapid Identification of Thrips palmi

    Journal: Frontiers in Molecular Biosciences

    doi: 10.3389/fmolb.2022.853339

    Visualization of PSR products using (A) . SYBR Green I, (B) . GoodView, and (C) . HNB. PSR was done using crude lysate of T. palmi as positive (+ve) and water as negative (-ve) control. Addition of 1 µl of SYBR Green I and GoodView after completion of PSR reaction showed fluorescence under UV light in positive samples, whereas no fluorescence was observed in negative samples. The PSR reaction was mixed with 2 µl of 3 mM HNB prior to amplification and showed a change in color from violet to sky blue in the case of T. palmi , while no corresponding color change was observed in negative samples.
    Figure Legend Snippet: Visualization of PSR products using (A) . SYBR Green I, (B) . GoodView, and (C) . HNB. PSR was done using crude lysate of T. palmi as positive (+ve) and water as negative (-ve) control. Addition of 1 µl of SYBR Green I and GoodView after completion of PSR reaction showed fluorescence under UV light in positive samples, whereas no fluorescence was observed in negative samples. The PSR reaction was mixed with 2 µl of 3 mM HNB prior to amplification and showed a change in color from violet to sky blue in the case of T. palmi , while no corresponding color change was observed in negative samples.

    Techniques Used: SYBR Green Assay, Fluorescence, Amplification

    Workflow of PSR assay for rapid, on-site detection of T. palmi . (A) . Single adult T. palmi collected from a leaf using a Camel hairbrush and placed in a 1.5 mlmicrocentrifuge tube, (B) . Thrips crushed with the help of a micro-pestle in sterile distilled water, (C) . Micro-centrifuge tube placed in a floating rack and incubated at 100°C in a water bath for 2 min, (D) . PSR mixture prepared in 0.5 ml PCR tubes, (E) . PSR reaction incubated in a water bath at 65°C for 60 min. Visual detection of presence or absence of T. palmi using (F) . SYBR Green I, (G) . GoodView. Fluorescence was observed in the positive samples while no fluorescence was detected in negative samples, (H) . Visual detection of T. palmi PSR products using HNB. Change in color from violet to sky blue indicates the presence of T. palmi , while no color change was observed in negative samples.
    Figure Legend Snippet: Workflow of PSR assay for rapid, on-site detection of T. palmi . (A) . Single adult T. palmi collected from a leaf using a Camel hairbrush and placed in a 1.5 mlmicrocentrifuge tube, (B) . Thrips crushed with the help of a micro-pestle in sterile distilled water, (C) . Micro-centrifuge tube placed in a floating rack and incubated at 100°C in a water bath for 2 min, (D) . PSR mixture prepared in 0.5 ml PCR tubes, (E) . PSR reaction incubated in a water bath at 65°C for 60 min. Visual detection of presence or absence of T. palmi using (F) . SYBR Green I, (G) . GoodView. Fluorescence was observed in the positive samples while no fluorescence was detected in negative samples, (H) . Visual detection of T. palmi PSR products using HNB. Change in color from violet to sky blue indicates the presence of T. palmi , while no color change was observed in negative samples.

    Techniques Used: Incubation, Polymerase Chain Reaction, SYBR Green Assay, Fluorescence

    4) Product Images from "Vpx mediated degradation of SAMHD1 has only a very limited effect on lentiviral transduction rate in ex vivo cultured HSPCs"

    Article Title: Vpx mediated degradation of SAMHD1 has only a very limited effect on lentiviral transduction rate in ex vivo cultured HSPCs

    Journal: Stem cell research

    doi: 10.1016/j.scr.2015.06.012

    Vpx+ virus, dN and dNTP treatments result in minor increases in the dNTP pool of HSPCs. MDMs (A, C and E) and freshly isolated HSPCs (B, D and F) were pretreated for 2 h with Vpx+/− viruses, dNs or dNTPs, and cultured for 3 days before cell lysis. Quantification of dNTPs was done by a single nucleotide incorporation assay (see “Material and methods” section). Fold induction was calculated based on the absolute values of vero- vs mock-treated samples (C–F). White dots represent Vpx− virus-pretreated samples, black dots represent Vpx+ virus-pretreated samples (A–B). White bars represent dN-pretreated samples, black bars represent dNTP-pretreated samples (C–F). Data are from three donors and bars represent mean ± SEM. Statistical analysis was done using two-tailed paired t-test.
    Figure Legend Snippet: Vpx+ virus, dN and dNTP treatments result in minor increases in the dNTP pool of HSPCs. MDMs (A, C and E) and freshly isolated HSPCs (B, D and F) were pretreated for 2 h with Vpx+/− viruses, dNs or dNTPs, and cultured for 3 days before cell lysis. Quantification of dNTPs was done by a single nucleotide incorporation assay (see “Material and methods” section). Fold induction was calculated based on the absolute values of vero- vs mock-treated samples (C–F). White dots represent Vpx− virus-pretreated samples, black dots represent Vpx+ virus-pretreated samples (A–B). White bars represent dN-pretreated samples, black bars represent dNTP-pretreated samples (C–F). Data are from three donors and bars represent mean ± SEM. Statistical analysis was done using two-tailed paired t-test.

    Techniques Used: Isolation, Cell Culture, Lysis, Two Tailed Test

    5) Product Images from "OverFlap PCR – a reliable approach for generation of plasmid DNA libraries containing random sequences without template bias"

    Article Title: OverFlap PCR – a reliable approach for generation of plasmid DNA libraries containing random sequences without template bias

    Journal: bioRxiv

    doi: 10.1101/2022.01.11.475799

    The principal scheme of proposed future OverFlap whole plasmid amplification based randomization of selected plasmid DNA region. Unlike in current iteration here the plasmid is linearized employing whole plasmid amplification and circularization is performed employing Gibson assembly. As an optional strategy for control of unwanted template circularization, one can use 1) dNTP mix with dUTP instead of dTTP and uridine tolerant high fidelity polymerase during template linearization to generate uridine containing template, 2) uridine tolerant high fidelity polymerase during asymmetric PCR and 3) USER enzyme mix (Uracil-Specific Excision Reagent) to selectively cleave template DNA. Randomized region is represented by red line; the template DNA is represented by black line; the synthesized chain is represented by grey line; Dotted line represents DNA synthesis.
    Figure Legend Snippet: The principal scheme of proposed future OverFlap whole plasmid amplification based randomization of selected plasmid DNA region. Unlike in current iteration here the plasmid is linearized employing whole plasmid amplification and circularization is performed employing Gibson assembly. As an optional strategy for control of unwanted template circularization, one can use 1) dNTP mix with dUTP instead of dTTP and uridine tolerant high fidelity polymerase during template linearization to generate uridine containing template, 2) uridine tolerant high fidelity polymerase during asymmetric PCR and 3) USER enzyme mix (Uracil-Specific Excision Reagent) to selectively cleave template DNA. Randomized region is represented by red line; the template DNA is represented by black line; the synthesized chain is represented by grey line; Dotted line represents DNA synthesis.

    Techniques Used: Plasmid Preparation, Amplification, Polymerase Chain Reaction, Synthesized, DNA Synthesis

    6) Product Images from "Seminal plasma induces inflammation and enhances HIV-1 replication in human cervical tissue explants"

    Article Title: Seminal plasma induces inflammation and enhances HIV-1 replication in human cervical tissue explants

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006402

    Infection of ectocervical tissue explants with HIV-1 BaL . Infection of ectocervical explants with HIV-1 BaL was independently performed after seminal plasma (SP) 25% treatment (post-SP,red), or in the presence of SP25% (SP-mix, blue) (see S1B Fig ). In selected experiments, explants infected in the presence of SP were treated with lamivudine (3TC) 10μM throughout culture time. A) Kinetics of HIV-1 BaL replication in explants treated with SP (colored line) or culture medium (CM) (black line). Virus replication was evaluated as p24 gag concentration in explant culture medium over 18 days. Represented are mean values with s.e.m. (n = 9 for post-SP; n = 8 for SP-mix; n = 3 for SP-mix+3TC). B) Cumulative p24 gag production over culture time. Lines connect measurements obtained from donor-matched explants. C) N-fold change in cumulative p24 gag production in SP-treated explants compared to donor-matched untreated explants. Bars indicate median values. p
    Figure Legend Snippet: Infection of ectocervical tissue explants with HIV-1 BaL . Infection of ectocervical explants with HIV-1 BaL was independently performed after seminal plasma (SP) 25% treatment (post-SP,red), or in the presence of SP25% (SP-mix, blue) (see S1B Fig ). In selected experiments, explants infected in the presence of SP were treated with lamivudine (3TC) 10μM throughout culture time. A) Kinetics of HIV-1 BaL replication in explants treated with SP (colored line) or culture medium (CM) (black line). Virus replication was evaluated as p24 gag concentration in explant culture medium over 18 days. Represented are mean values with s.e.m. (n = 9 for post-SP; n = 8 for SP-mix; n = 3 for SP-mix+3TC). B) Cumulative p24 gag production over culture time. Lines connect measurements obtained from donor-matched explants. C) N-fold change in cumulative p24 gag production in SP-treated explants compared to donor-matched untreated explants. Bars indicate median values. p

    Techniques Used: Infection, Concentration Assay

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher dntp
    Evaluation of direct cell lysis protocols on RT-qPCR . (A) The RT-qPCR yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike using 17 lysis conditions. Five nanograms of purified RNA was used in all RT reactions. Relative RT yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage relative to the water control for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Lysis conditions with Cq-values below that of the water control are RT enhancing agents, while conditions with higher Cq-values are inhibitory. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are shown in Table S4 in Supplementary Material. (B) Mean RT yield for Gapdh , Vim , Dll , and Jag1 . The relative transcript yield of each transcript was averaged and compared to the optimal RT-qPCR condition (RT mix). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM <t>oligo-dT,</t> and 1 mM <t>dNTP.</t>
    Dntp, 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
    https://www.bioz.com/result/dntp/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dntp - by Bioz Stars, 2022-08
    99/100 stars
      Buy from Supplier

    Image Search Results


    Evaluation of direct cell lysis protocols on RT-qPCR . (A) The RT-qPCR yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike using 17 lysis conditions. Five nanograms of purified RNA was used in all RT reactions. Relative RT yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage relative to the water control for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Lysis conditions with Cq-values below that of the water control are RT enhancing agents, while conditions with higher Cq-values are inhibitory. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are shown in Table S4 in Supplementary Material. (B) Mean RT yield for Gapdh , Vim , Dll , and Jag1 . The relative transcript yield of each transcript was averaged and compared to the optimal RT-qPCR condition (RT mix). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

    Journal: Frontiers in Oncology

    Article Title: Direct Cell Lysis for Single-Cell Gene Expression Profiling

    doi: 10.3389/fonc.2013.00274

    Figure Lengend Snippet: Evaluation of direct cell lysis protocols on RT-qPCR . (A) The RT-qPCR yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike using 17 lysis conditions. Five nanograms of purified RNA was used in all RT reactions. Relative RT yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage relative to the water control for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Lysis conditions with Cq-values below that of the water control are RT enhancing agents, while conditions with higher Cq-values are inhibitory. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are shown in Table S4 in Supplementary Material. (B) Mean RT yield for Gapdh , Vim , Dll , and Jag1 . The relative transcript yield of each transcript was averaged and compared to the optimal RT-qPCR condition (RT mix). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

    Article Snippet: The following chemicals were evaluated (final lysis concentrations are shown): 7-deaza-2′-deoxyguanosine-5′-triphosphate lithium salt (100 μM, Sigma-Aldrich); Betaine solution (4 M, Sigma-Aldrich); BSA (1–4 mg/ml, Fermentas); guanidine thiocyanate solution (GTC) (40–80 mM, Sigma-Aldrich); GenElute linear polyacrylamide (LPA) (50 ng/μl, Sigma-Aldrich); Igepal CA-630 (also known as Non-idet P-40, 0.5–4%, Sigma-Aldrich); polyinosinic acid potassium salt (50 ng/μl, Sigma-Aldrich); RNAseOUT (10 U/μl, Invitrogen); 2× reverse transcription buffer: 100 mM Tris-HCl (pH 8.3), 150 mM KCl, and 6 mM MgCl2 (Invitrogen); d -(+)-trehalose dihydrate (1 M, Sigma-Aldrich); yeast tRNA (50 ng/μl, Ambion); RT mix (2× RT buffer, Invitrogen, 5 μM random hexamers (Metabion), 5 μM oligo-dT (Metabion), 1 mM dNTP); RT mix with BSA (2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, 1 mM dNTP, 1 mg/ml BSA); and RNase-free water (Gibco).

    Techniques: Lysis, Quantitative RT-PCR, Purification, Polymerase Chain Reaction

    Evaluation of direct cell lysis protocols . (A) The lysis yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike compared at 17 lysis conditions. Thirty-two astrocytes were sorted for each condition. Relative cDNA yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage compared to the optimal lysis condition for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are listed in Table S3 in Supplementary Material. (B) Mean cDNA yield of the transcripts. Expressions of Gapdh , Vim , Dll , and Jag1 were averaged and are compared to the overall optimal lysis condition (1 mg/ml BSA). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

    Journal: Frontiers in Oncology

    Article Title: Direct Cell Lysis for Single-Cell Gene Expression Profiling

    doi: 10.3389/fonc.2013.00274

    Figure Lengend Snippet: Evaluation of direct cell lysis protocols . (A) The lysis yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike compared at 17 lysis conditions. Thirty-two astrocytes were sorted for each condition. Relative cDNA yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage compared to the optimal lysis condition for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are listed in Table S3 in Supplementary Material. (B) Mean cDNA yield of the transcripts. Expressions of Gapdh , Vim , Dll , and Jag1 were averaged and are compared to the overall optimal lysis condition (1 mg/ml BSA). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

    Article Snippet: The following chemicals were evaluated (final lysis concentrations are shown): 7-deaza-2′-deoxyguanosine-5′-triphosphate lithium salt (100 μM, Sigma-Aldrich); Betaine solution (4 M, Sigma-Aldrich); BSA (1–4 mg/ml, Fermentas); guanidine thiocyanate solution (GTC) (40–80 mM, Sigma-Aldrich); GenElute linear polyacrylamide (LPA) (50 ng/μl, Sigma-Aldrich); Igepal CA-630 (also known as Non-idet P-40, 0.5–4%, Sigma-Aldrich); polyinosinic acid potassium salt (50 ng/μl, Sigma-Aldrich); RNAseOUT (10 U/μl, Invitrogen); 2× reverse transcription buffer: 100 mM Tris-HCl (pH 8.3), 150 mM KCl, and 6 mM MgCl2 (Invitrogen); d -(+)-trehalose dihydrate (1 M, Sigma-Aldrich); yeast tRNA (50 ng/μl, Ambion); RT mix (2× RT buffer, Invitrogen, 5 μM random hexamers (Metabion), 5 μM oligo-dT (Metabion), 1 mM dNTP); RT mix with BSA (2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, 1 mM dNTP, 1 mg/ml BSA); and RNase-free water (Gibco).

    Techniques: Lysis, Polymerase Chain Reaction

    Visualization of PSR products using (A) . SYBR Green I, (B) . GoodView, and (C) . HNB. PSR was done using crude lysate of T. palmi as positive (+ve) and water as negative (-ve) control. Addition of 1 µl of SYBR Green I and GoodView after completion of PSR reaction showed fluorescence under UV light in positive samples, whereas no fluorescence was observed in negative samples. The PSR reaction was mixed with 2 µl of 3 mM HNB prior to amplification and showed a change in color from violet to sky blue in the case of T. palmi , while no corresponding color change was observed in negative samples.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Development of a Polymerase Spiral Reaction-Based Isothermal Assay for Rapid Identification of Thrips palmi

    doi: 10.3389/fmolb.2022.853339

    Figure Lengend Snippet: Visualization of PSR products using (A) . SYBR Green I, (B) . GoodView, and (C) . HNB. PSR was done using crude lysate of T. palmi as positive (+ve) and water as negative (-ve) control. Addition of 1 µl of SYBR Green I and GoodView after completion of PSR reaction showed fluorescence under UV light in positive samples, whereas no fluorescence was observed in negative samples. The PSR reaction was mixed with 2 µl of 3 mM HNB prior to amplification and showed a change in color from violet to sky blue in the case of T. palmi , while no corresponding color change was observed in negative samples.

    Article Snippet: The optimized PSR reaction mixture was comprised of 2.5 µl of 10X Thermopol reaction buffer, 40 µM each forward and reverse primer, 1.4 mM dNTP mix, 0.8 M Betaine, 12 mM MgSO4, 16 U of Bst DNA polymerase large fragment, 50 ng DNA template, and the final volume was adjusted to 25 µl with sterile distilled water.

    Techniques: SYBR Green Assay, Fluorescence, Amplification

    Workflow of PSR assay for rapid, on-site detection of T. palmi . (A) . Single adult T. palmi collected from a leaf using a Camel hairbrush and placed in a 1.5 mlmicrocentrifuge tube, (B) . Thrips crushed with the help of a micro-pestle in sterile distilled water, (C) . Micro-centrifuge tube placed in a floating rack and incubated at 100°C in a water bath for 2 min, (D) . PSR mixture prepared in 0.5 ml PCR tubes, (E) . PSR reaction incubated in a water bath at 65°C for 60 min. Visual detection of presence or absence of T. palmi using (F) . SYBR Green I, (G) . GoodView. Fluorescence was observed in the positive samples while no fluorescence was detected in negative samples, (H) . Visual detection of T. palmi PSR products using HNB. Change in color from violet to sky blue indicates the presence of T. palmi , while no color change was observed in negative samples.

    Journal: Frontiers in Molecular Biosciences

    Article Title: Development of a Polymerase Spiral Reaction-Based Isothermal Assay for Rapid Identification of Thrips palmi

    doi: 10.3389/fmolb.2022.853339

    Figure Lengend Snippet: Workflow of PSR assay for rapid, on-site detection of T. palmi . (A) . Single adult T. palmi collected from a leaf using a Camel hairbrush and placed in a 1.5 mlmicrocentrifuge tube, (B) . Thrips crushed with the help of a micro-pestle in sterile distilled water, (C) . Micro-centrifuge tube placed in a floating rack and incubated at 100°C in a water bath for 2 min, (D) . PSR mixture prepared in 0.5 ml PCR tubes, (E) . PSR reaction incubated in a water bath at 65°C for 60 min. Visual detection of presence or absence of T. palmi using (F) . SYBR Green I, (G) . GoodView. Fluorescence was observed in the positive samples while no fluorescence was detected in negative samples, (H) . Visual detection of T. palmi PSR products using HNB. Change in color from violet to sky blue indicates the presence of T. palmi , while no color change was observed in negative samples.

    Article Snippet: The optimized PSR reaction mixture was comprised of 2.5 µl of 10X Thermopol reaction buffer, 40 µM each forward and reverse primer, 1.4 mM dNTP mix, 0.8 M Betaine, 12 mM MgSO4, 16 U of Bst DNA polymerase large fragment, 50 ng DNA template, and the final volume was adjusted to 25 µl with sterile distilled water.

    Techniques: Incubation, Polymerase Chain Reaction, SYBR Green Assay, Fluorescence

    Creating a supercoiled and fluorescent-labeled sopC-plasmid. The plasmid pBR322:: sopC is fluorescently labeled to visualize its movement over the DNA-carpeted flow cell. We have developed an efficient labeling protocol that does not require intercalating dyes and produces a negatively supercoiled plasmid. The restriction enzyme Nt.BspQ1 nicks the pBR322 backbone at a site located approximately 180° from sopC . DNA polymerase I is used with dNTPs and Alexa647-labeled dCTP to label the DNA. Ethidium Bromide promotes negative supercoiling before a final ligation reaction that covalently closes the nick. The final product is a negatively supercoiled and fluorescently labeled plasmid bearing the sopC centromere site. This protocol can be used to incorporate a variety of dyes without significant perturbation to plasmid topology.

    Journal: Methods in cell biology

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo

    doi: 10.1016/bs.mcb.2015.01.021

    Figure Lengend Snippet: Creating a supercoiled and fluorescent-labeled sopC-plasmid. The plasmid pBR322:: sopC is fluorescently labeled to visualize its movement over the DNA-carpeted flow cell. We have developed an efficient labeling protocol that does not require intercalating dyes and produces a negatively supercoiled plasmid. The restriction enzyme Nt.BspQ1 nicks the pBR322 backbone at a site located approximately 180° from sopC . DNA polymerase I is used with dNTPs and Alexa647-labeled dCTP to label the DNA. Ethidium Bromide promotes negative supercoiling before a final ligation reaction that covalently closes the nick. The final product is a negatively supercoiled and fluorescently labeled plasmid bearing the sopC centromere site. This protocol can be used to incorporate a variety of dyes without significant perturbation to plasmid topology.

    Article Snippet: pBR322:: sopC plasmid (PCR template) PCR Primers for sopC DNA amplification: (Primers synthesized by IDT) 10 mM dNTPs (Life Technologies; Cat. # 18427–013) 100 mM MgSO4 (New England Biolabs, Cat. # M0254S) 2000 U/mL VENT Polymerase (New England Biolabs, Cat. # M0254S) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) MyOne Streptavidin C1 Dynabeads (Life Technologies, Cat. # 65001) 20 U/μL PstI Restriction Enzyme (New England Biolabs, Cat. # R0140S) Phenol:CHCl3:Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol.

    Techniques: Labeling, Plasmid Preparation, Flow Cytometry, Ligation