sybr select master mix  (Thermo Fisher)


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    Name:
    SYBR Safe DNA Gel Stain
    Description:
    SYBR Safe DNA Gel Stain is a highly sensitive stain for visualization of DNA in agarose or acrylamide gels. SYBR Safe stain is specifically formulated to be a less hazardous alternative to ethidium bromide that can utilize either blue light or UV excitation.• Reduce your exposure to highly mutagenic ethidium bromide and harmful UV light• Increase your sensitivity by reducing nonspecific background fluorescence• Use in place of ethidium bromide for all staining applications, including RNA stainingA better DNA stainSYBR Safe DNA Gel Stain is a better nucleic acid staining reagent for all of your molecular biology needs. Not only is SYBR Safe DNA Gel Stain better for you and the environment, it’s better for your sample and your institution. Read more as to why SYBR Safe DNA gel stain is your better option.Safer than ethidium bromideSYBR Safe DNA Gel Stain has been specifically formulated and evaluated in a battery of toxicity tests with results indicating so that it is less mutagenic and safer for you to work with than ethidium bromide. An independent laboratory showed reduced mutagenicity of SYBR Safe stain compared to ethidium bromide using the Ames test (see figure). You can further decrease your exposure risk by using visible blue-light illumination with SYBR Safe stain rather than UV illumination. This is especially valuable when performing exposure-intensive protocols like cutting bands out of gels.Excellent sensitivitySYBR Safe stain offers excellent sensitivity in nucleic acid visualization and documentation applications with either UV excitation or blue-light excitation. When bound to nucleic acids, the green-fluorescent SYBR Safe stain has fluorescence excitation maxima at ~280 and ~502 nm, and an emission maximum at ~530 nm (see figure). Plus, when used with blue light illumination, SYBR Safe stain has less background fluorescence than ethidium bromide–stained gels illuminated with UV light.Easy to useSYBR Safe stain is supplied as a 10,000X concentrate in DMSO which can be used just like a solution of ethidium bromide. SYBR Safe stain can be mixed into an agarose gel for staining during electrophoresis or the gel can be incubated in a solution of SYBR Safe stain following electrophoresis. SYBR Safe stain can be stored at room temperature in its original packaging to avoid excessive light exposure. We also offer SYBR Safe E-Gel pre-cast agarose gels for the ultimate in convenience.
    Catalog Number:
    S33102
    Price:
    None
    Applications:
    Agarose Gel Electrophoresis|DNA & RNA Purification & Analysis|Pour Your Own Agarose Gels|Nucleic Acid Gel Electrophoresis & Blotting
    Conjugate:
    SYBR Safe
    Size:
    400 µL
    Category:
    Labeling & Detection Products, Gel & Membrane Stains
    Score:
    85
    Buy from Supplier


    Structured Review

    Thermo Fisher sybr select master mix
    Not all <t>PCR</t> mixes result in optimal specificity of cDNA amplification reactions. The performance of three commercial <t>SYBR</t> green PCR mixes was compared by amplifying cDNA with 70 primer-based assays targeting different mRNA transcripts. A single melting curve peak indicates specificity of the amplification. The figure shows examples of melting curves and corresponding gel blots for several primer sets from Table 3 , in situations where all three mixes gave optimal results and where one or more mixes resulted in a suboptimal amplification reaction
    SYBR Safe DNA Gel Stain is a highly sensitive stain for visualization of DNA in agarose or acrylamide gels. SYBR Safe stain is specifically formulated to be a less hazardous alternative to ethidium bromide that can utilize either blue light or UV excitation.• Reduce your exposure to highly mutagenic ethidium bromide and harmful UV light• Increase your sensitivity by reducing nonspecific background fluorescence• Use in place of ethidium bromide for all staining applications, including RNA stainingA better DNA stainSYBR Safe DNA Gel Stain is a better nucleic acid staining reagent for all of your molecular biology needs. Not only is SYBR Safe DNA Gel Stain better for you and the environment, it’s better for your sample and your institution. Read more as to why SYBR Safe DNA gel stain is your better option.Safer than ethidium bromideSYBR Safe DNA Gel Stain has been specifically formulated and evaluated in a battery of toxicity tests with results indicating so that it is less mutagenic and safer for you to work with than ethidium bromide. An independent laboratory showed reduced mutagenicity of SYBR Safe stain compared to ethidium bromide using the Ames test (see figure). You can further decrease your exposure risk by using visible blue-light illumination with SYBR Safe stain rather than UV illumination. This is especially valuable when performing exposure-intensive protocols like cutting bands out of gels.Excellent sensitivitySYBR Safe stain offers excellent sensitivity in nucleic acid visualization and documentation applications with either UV excitation or blue-light excitation. When bound to nucleic acids, the green-fluorescent SYBR Safe stain has fluorescence excitation maxima at ~280 and ~502 nm, and an emission maximum at ~530 nm (see figure). Plus, when used with blue light illumination, SYBR Safe stain has less background fluorescence than ethidium bromide–stained gels illuminated with UV light.Easy to useSYBR Safe stain is supplied as a 10,000X concentrate in DMSO which can be used just like a solution of ethidium bromide. SYBR Safe stain can be mixed into an agarose gel for staining during electrophoresis or the gel can be incubated in a solution of SYBR Safe stain following electrophoresis. SYBR Safe stain can be stored at room temperature in its original packaging to avoid excessive light exposure. We also offer SYBR Safe E-Gel pre-cast agarose gels for the ultimate in convenience.
    https://www.bioz.com/result/sybr select master mix/product/Thermo Fisher
    Average 99 stars, based on 503 article reviews
    Price from $9.99 to $1999.99
    sybr select master mix - by Bioz Stars, 2020-01
    99/100 stars

    Images

    1) Product Images from "The source of SYBR green master mix determines outcome of nucleic acid amplification reactions"

    Article Title: The source of SYBR green master mix determines outcome of nucleic acid amplification reactions

    Journal: BMC Research Notes

    doi: 10.1186/s13104-016-2093-4

    Not all PCR mixes result in optimal specificity of cDNA amplification reactions. The performance of three commercial SYBR green PCR mixes was compared by amplifying cDNA with 70 primer-based assays targeting different mRNA transcripts. A single melting curve peak indicates specificity of the amplification. The figure shows examples of melting curves and corresponding gel blots for several primer sets from Table 3 , in situations where all three mixes gave optimal results and where one or more mixes resulted in a suboptimal amplification reaction
    Figure Legend Snippet: Not all PCR mixes result in optimal specificity of cDNA amplification reactions. The performance of three commercial SYBR green PCR mixes was compared by amplifying cDNA with 70 primer-based assays targeting different mRNA transcripts. A single melting curve peak indicates specificity of the amplification. The figure shows examples of melting curves and corresponding gel blots for several primer sets from Table 3 , in situations where all three mixes gave optimal results and where one or more mixes resulted in a suboptimal amplification reaction

    Techniques Used: Polymerase Chain Reaction, Amplification, SYBR Green Assay

    2) Product Images from "Disruption of the Zdhhc9 intellectual disability gene leads to behavioural abnormalities in a mouse model"

    Article Title: Disruption of the Zdhhc9 intellectual disability gene leads to behavioural abnormalities in a mouse model

    Journal: Experimental Neurology

    doi: 10.1016/j.expneurol.2018.06.014

    PCR and immunoblotting analysis of Zdhhc9 expression in wild-type and mutant mice. (A) Agarose gel electrophoresis of end-point PCR products from WT and mutant brain cDNA samples. mRNA confirmation assay 1 (MA1) primers were designed to amplify a Zdhhc9 mRNA region of 283 bp from the 5′UTR to Exon 2. mRNA confirmation assay 2 (MA2) primers were designed to amplify a Zdhhc9 mRNA region of 154 bp from Exon 2 to Exon 3. z9v1 primers were designed to amplify a mRNA region of 139 bp from exon 5 to exon 7, and z9v2 primers were designed to amplify a mRNA region of 106 bp from exon 5 to exon 7. HyperLadder 100 bp ladder (Bioline) was used as a marker of DNA size. (B) Comparison of the average ΔCt values of WT and mutant mouse brain samples ( n = 3 WT, 3 mutant) for MA2 and z9v1 after normalisation against Tbp and Hprt1 reference genes. cDNA from WT and mutant mouse brain was amplified for 40 cycles using specific primers for the different targets (MA2, z9v1, Tbp and Hprt1 ) and SYBR Select Master Mix. Statistical analysis (unpaired t -test, Minitab version 17) revealed a significant effect of genotype for MA2- Tbp ( p = .042), MA2- Hprt1 ( p = .046) and z9v1- Hprt1 ( p = .037). p value for z9v1- Tbp was 0.075. (C) PCR products amplified from cDNA derived from mRNA extracted from WT and Zdhhc9 mutant mouse brain. Primers were designed to anneal to a region in exon 1 (see MAO F in for sequence of primer) and the 3′-UTR (3UTR R in for sequence of primer). HyperLadder 1 kb ladder (Bioline) was included as marker of DNA size. (D) Lysates from HEK293T cells transfected with HA-tagged zDHHCs were probed with antibodies against zDHHC9 ( top ) and HA ( bottom ). (E) Brain lysates from WT and mutant mice were probed with antibodies against zDHHC9 ( top ) and beta actin ( bottom ). Position of molecular weight markers is shown on the left of all immunoblots.
    Figure Legend Snippet: PCR and immunoblotting analysis of Zdhhc9 expression in wild-type and mutant mice. (A) Agarose gel electrophoresis of end-point PCR products from WT and mutant brain cDNA samples. mRNA confirmation assay 1 (MA1) primers were designed to amplify a Zdhhc9 mRNA region of 283 bp from the 5′UTR to Exon 2. mRNA confirmation assay 2 (MA2) primers were designed to amplify a Zdhhc9 mRNA region of 154 bp from Exon 2 to Exon 3. z9v1 primers were designed to amplify a mRNA region of 139 bp from exon 5 to exon 7, and z9v2 primers were designed to amplify a mRNA region of 106 bp from exon 5 to exon 7. HyperLadder 100 bp ladder (Bioline) was used as a marker of DNA size. (B) Comparison of the average ΔCt values of WT and mutant mouse brain samples ( n = 3 WT, 3 mutant) for MA2 and z9v1 after normalisation against Tbp and Hprt1 reference genes. cDNA from WT and mutant mouse brain was amplified for 40 cycles using specific primers for the different targets (MA2, z9v1, Tbp and Hprt1 ) and SYBR Select Master Mix. Statistical analysis (unpaired t -test, Minitab version 17) revealed a significant effect of genotype for MA2- Tbp ( p = .042), MA2- Hprt1 ( p = .046) and z9v1- Hprt1 ( p = .037). p value for z9v1- Tbp was 0.075. (C) PCR products amplified from cDNA derived from mRNA extracted from WT and Zdhhc9 mutant mouse brain. Primers were designed to anneal to a region in exon 1 (see MAO F in for sequence of primer) and the 3′-UTR (3UTR R in for sequence of primer). HyperLadder 1 kb ladder (Bioline) was included as marker of DNA size. (D) Lysates from HEK293T cells transfected with HA-tagged zDHHCs were probed with antibodies against zDHHC9 ( top ) and HA ( bottom ). (E) Brain lysates from WT and mutant mice were probed with antibodies against zDHHC9 ( top ) and beta actin ( bottom ). Position of molecular weight markers is shown on the left of all immunoblots.

    Techniques Used: Polymerase Chain Reaction, Expressing, Mutagenesis, Mouse Assay, Agarose Gel Electrophoresis, Marker, Amplification, Derivative Assay, Sequencing, Transfection, Hemagglutination Assay, Molecular Weight, Western Blot

    3) Product Images from "Regulation of matrix metalloproteinases (MMPs) expression and secretion in MDA-MB-231 breast cancer cells by LIM and SH3 protein 1 (LASP1)"

    Article Title: Regulation of matrix metalloproteinases (MMPs) expression and secretion in MDA-MB-231 breast cancer cells by LIM and SH3 protein 1 (LASP1)

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11720

    Loss of LASP1 in breast cancer cells affects gene expression of matrix metalloproteinases qRT-PCR in human MDA-MB-231-shLASP1 cells was performed with SYBR Select Master Mix. Bar graphs show the relative mRNA expression, calculated by the comparative ddCT-method and normalized to the housekeeping gene ribosomalprotein large P0 (RPLP0). LASP1 depletion decreased MMP1, MMP3, and MMP9 gene expression while MMP2 gene expression increased. MMP14 mRNA was not affected by LASP1 knockdown. *p
    Figure Legend Snippet: Loss of LASP1 in breast cancer cells affects gene expression of matrix metalloproteinases qRT-PCR in human MDA-MB-231-shLASP1 cells was performed with SYBR Select Master Mix. Bar graphs show the relative mRNA expression, calculated by the comparative ddCT-method and normalized to the housekeeping gene ribosomalprotein large P0 (RPLP0). LASP1 depletion decreased MMP1, MMP3, and MMP9 gene expression while MMP2 gene expression increased. MMP14 mRNA was not affected by LASP1 knockdown. *p

    Techniques Used: Expressing, Quantitative RT-PCR, Multiple Displacement Amplification

    4) Product Images from "Disruption of the Zdhhc9 intellectual disability gene leads to behavioural abnormalities in a mouse model"

    Article Title: Disruption of the Zdhhc9 intellectual disability gene leads to behavioural abnormalities in a mouse model

    Journal: Experimental Neurology

    doi: 10.1016/j.expneurol.2018.06.014

    PCR and immunoblotting analysis of Zdhhc9 expression in wild-type and mutant mice. (A) Agarose gel electrophoresis of end-point PCR products from WT and mutant brain cDNA samples. mRNA confirmation assay 1 (MA1) primers were designed to amplify a Zdhhc9 mRNA region of 283 bp from the 5′UTR to Exon 2. mRNA confirmation assay 2 (MA2) primers were designed to amplify a Zdhhc9 mRNA region of 154 bp from Exon 2 to Exon 3. z9v1 primers were designed to amplify a mRNA region of 139 bp from exon 5 to exon 7, and z9v2 primers were designed to amplify a mRNA region of 106 bp from exon 5 to exon 7. HyperLadder 100 bp ladder (Bioline) was used as a marker of DNA size. (B) Comparison of the average ΔCt values of WT and mutant mouse brain samples ( n = 3 WT, 3 mutant) for MA2 and z9v1 after normalisation against Tbp and Hprt1 reference genes. cDNA from WT and mutant mouse brain was amplified for 40 cycles using specific primers for the different targets (MA2, z9v1, Tbp and Hprt1 ) and SYBR Select Master Mix. Statistical analysis (unpaired t -test, Minitab version 17) revealed a significant effect of genotype for MA2- Tbp ( p = .042), MA2- Hprt1 ( p = .046) and z9v1- Hprt1 ( p = .037). p value for z9v1- Tbp was 0.075. (C) PCR products amplified from cDNA derived from mRNA extracted from WT and Zdhhc9 mutant mouse brain. Primers were designed to anneal to a region in exon 1 (see MAO F in Table 1 for sequence of primer) and the 3′-UTR (3UTR R in Table 1 for sequence of primer). HyperLadder 1 kb ladder (Bioline) was included as marker of DNA size. (D) Lysates from HEK293T cells transfected with HA-tagged zDHHCs were probed with antibodies against zDHHC9 ( top ) and HA ( bottom ). (E) Brain lysates from WT and mutant mice were probed with antibodies against zDHHC9 ( top ) and beta actin ( bottom ). Position of molecular weight markers is shown on the left of all immunoblots.
    Figure Legend Snippet: PCR and immunoblotting analysis of Zdhhc9 expression in wild-type and mutant mice. (A) Agarose gel electrophoresis of end-point PCR products from WT and mutant brain cDNA samples. mRNA confirmation assay 1 (MA1) primers were designed to amplify a Zdhhc9 mRNA region of 283 bp from the 5′UTR to Exon 2. mRNA confirmation assay 2 (MA2) primers were designed to amplify a Zdhhc9 mRNA region of 154 bp from Exon 2 to Exon 3. z9v1 primers were designed to amplify a mRNA region of 139 bp from exon 5 to exon 7, and z9v2 primers were designed to amplify a mRNA region of 106 bp from exon 5 to exon 7. HyperLadder 100 bp ladder (Bioline) was used as a marker of DNA size. (B) Comparison of the average ΔCt values of WT and mutant mouse brain samples ( n = 3 WT, 3 mutant) for MA2 and z9v1 after normalisation against Tbp and Hprt1 reference genes. cDNA from WT and mutant mouse brain was amplified for 40 cycles using specific primers for the different targets (MA2, z9v1, Tbp and Hprt1 ) and SYBR Select Master Mix. Statistical analysis (unpaired t -test, Minitab version 17) revealed a significant effect of genotype for MA2- Tbp ( p = .042), MA2- Hprt1 ( p = .046) and z9v1- Hprt1 ( p = .037). p value for z9v1- Tbp was 0.075. (C) PCR products amplified from cDNA derived from mRNA extracted from WT and Zdhhc9 mutant mouse brain. Primers were designed to anneal to a region in exon 1 (see MAO F in Table 1 for sequence of primer) and the 3′-UTR (3UTR R in Table 1 for sequence of primer). HyperLadder 1 kb ladder (Bioline) was included as marker of DNA size. (D) Lysates from HEK293T cells transfected with HA-tagged zDHHCs were probed with antibodies against zDHHC9 ( top ) and HA ( bottom ). (E) Brain lysates from WT and mutant mice were probed with antibodies against zDHHC9 ( top ) and beta actin ( bottom ). Position of molecular weight markers is shown on the left of all immunoblots.

    Techniques Used: Polymerase Chain Reaction, Expressing, Mutagenesis, Mouse Assay, Agarose Gel Electrophoresis, Marker, Amplification, Derivative Assay, Sequencing, Transfection, Hemagglutination Assay, Molecular Weight, Western Blot

    5) Product Images from "The ESCRT-Related ATPase Vps4 Is Modulated by Interferon during Herpes Simplex Virus 1 Infection"

    Article Title: The ESCRT-Related ATPase Vps4 Is Modulated by Interferon during Herpes Simplex Virus 1 Infection

    Journal: mBio

    doi: 10.1128/mBio.02567-18

    IFNs decrease Vps4A and Vps4B mRNA levels in TG mouse neurons. (A) mRNA levels of Vps4A, Vps4B, Chmp4B, Chmp2B, and Isg15 were analyzed by qPCR using TG mRNA from infected LC3-GFP +/− mice during the indicated times. Quantification of Sybr Green signal was normalized to β-actin; n = 5 for all genes analyzed and all time points, except for Chmp2B Mock ( n = 3). All samples were performed with two technical replicates. *, P
    Figure Legend Snippet: IFNs decrease Vps4A and Vps4B mRNA levels in TG mouse neurons. (A) mRNA levels of Vps4A, Vps4B, Chmp4B, Chmp2B, and Isg15 were analyzed by qPCR using TG mRNA from infected LC3-GFP +/− mice during the indicated times. Quantification of Sybr Green signal was normalized to β-actin; n = 5 for all genes analyzed and all time points, except for Chmp2B Mock ( n = 3). All samples were performed with two technical replicates. *, P

    Techniques Used: Real-time Polymerase Chain Reaction, Infection, Mouse Assay, SYBR Green Assay

    6) Product Images from "Oncogenic H-Ras Expression Induces Fatty Acid Profile Changes in Human Fibroblasts and Extracellular Vesicles"

    Article Title: Oncogenic H-Ras Expression Induces Fatty Acid Profile Changes in Human Fibroblasts and Extracellular Vesicles

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19113515

    Gene expression analysis of fatty acid-metabolizing enzymes in control and H-RasV12 expressing fibroblasts by qRT-PCR. ( A ) Gene expression analysis of desaturases ( SCD and FADS ), elongases ( ELOVL ) and acyl-coenzyme A synthetases ( ACSL ). ( B ) Gene expression analysis of phospholipases A2 ( PLA2 ). Ten ng of each cDNA were used as template. Reactions were performed in triplicate, using SYBR green technology StepOne RT-PCR machine to detect amplification. The GAPDH gene was used as endogenous control. The fold expression in H-RasV12 fibroblasts with respect to control is displayed, expressed as Relative Quantity (RQ). The analysis was repeated three times in triplicate and the mean ± S.D. is reported (* p
    Figure Legend Snippet: Gene expression analysis of fatty acid-metabolizing enzymes in control and H-RasV12 expressing fibroblasts by qRT-PCR. ( A ) Gene expression analysis of desaturases ( SCD and FADS ), elongases ( ELOVL ) and acyl-coenzyme A synthetases ( ACSL ). ( B ) Gene expression analysis of phospholipases A2 ( PLA2 ). Ten ng of each cDNA were used as template. Reactions were performed in triplicate, using SYBR green technology StepOne RT-PCR machine to detect amplification. The GAPDH gene was used as endogenous control. The fold expression in H-RasV12 fibroblasts with respect to control is displayed, expressed as Relative Quantity (RQ). The analysis was repeated three times in triplicate and the mean ± S.D. is reported (* p

    Techniques Used: Expressing, Quantitative RT-PCR, SYBR Green Assay, Step One RT-PCR, Amplification

    Related Articles

    Diagnostic Assay:

    Article Title: Raman Microspectroscopy Analysis in the Treatment of Acanthamoeba Keratitis
    Article Snippet: A 405-bp region of the 18S-rRNA gene (ASA.S1) that includes the Diagnostic Fragment 3 (DF3) was amplified using the genus-specific primers JDP1 and JDP2 to a final volume of 25 µ l. The PCR program was as follows: denaturing step at 96°C for 2 min, followed by 35 cycles of denaturing for 1 min at 96°C, annealing for 1 min at 60°C and extension for 1 min at 72°C, followed by a final extension at 72°C for 7 min. Sterile distilled water was included as negative control in each batch of DNA extraction and PCR reactions. .. Bands were visualised on SYBR SafeTM DNA gel stain (Invitrogen Corporation, Italy) stained 1% agarose gels.

    Amplification:

    Article Title: Neuronal TRPV1 activation regulates alveolar bone resorption by suppressing osteoclastogenesis via CGRP
    Article Snippet: PCR products were run on 1.5% agarose gels and visualized using SYBR® Safe DNA (Invitrogen Corporation, Carlsbad, CA, USA). .. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh ) was amplified using forward primer 5′-TCAACAGCAACTCCCACTCTT-3′ and reverse primer 5′-ACCCTGTTGCTGTAGCCGTAT-3′.

    Article Title: Human papillomavirus, high-grade intraepithelial neoplasia and killer immunoglogulin-like receptors: a Western Australian cohort study
    Article Snippet: KIR genotyping was performed on all samples using multiplexed amplification primers specific for the following KIR genes: 2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3 and 3DS1 as described by Sun et al. [ ]. .. PCR products were electrophoresed on a 2% agarose gel in 1× TAE buffer precast in SYBR Safe (Invitrogen, Carlsbad, CA) and photographed under a UV transilluminator.

    Article Title: Serological and molecular epidemiology of canine adenovirus type 1 in red foxes (Vulpes vulpes) in the United Kingdom
    Article Snippet: PCR products were loaded on agarose gels stained with SYBR safe DNA stain (Invitrogen, Paisley, UK) and separated by electrophoresis. .. PCR products were loaded on agarose gels stained with SYBR safe DNA stain (Invitrogen, Paisley, UK) and separated by electrophoresis.

    Article Title: Structure, organization and tissue expression of the pig SLC13A1 and SLC13A4 sulfate transporter genes
    Article Snippet: Control 470 bp β-actin cDNA fragments were amplified using forward 5’- GGACCTGACCGACTACCTCA-3’ and reverse 5’-ACACGGAGTACTTGCGCTCT-3’ primer sequences. .. The thermal cycling protocol was: 35 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min; followed by 72 °C for 5 min. PCR-amplified DNA was size-fractioned in a 1.5% agarose gel and then visualised using SYBR® safe DNA stain (Invitrogen) under UV light.

    Article Title: Raman Microspectroscopy Analysis in the Treatment of Acanthamoeba Keratitis
    Article Snippet: A 405-bp region of the 18S-rRNA gene (ASA.S1) that includes the Diagnostic Fragment 3 (DF3) was amplified using the genus-specific primers JDP1 and JDP2 to a final volume of 25 µ l. The PCR program was as follows: denaturing step at 96°C for 2 min, followed by 35 cycles of denaturing for 1 min at 96°C, annealing for 1 min at 60°C and extension for 1 min at 72°C, followed by a final extension at 72°C for 7 min. Sterile distilled water was included as negative control in each batch of DNA extraction and PCR reactions. .. Bands were visualised on SYBR SafeTM DNA gel stain (Invitrogen Corporation, Italy) stained 1% agarose gels.

    Article Title: Chromatin Immunoprecipitation (ChIP) using Drosophila tissue
    Article Snippet: For each primer pair, set up qPCR in duplicate in a 96-well plate with genomic DNA from step 3a.1, input control and ChIP-ed DNA:10 μL 2x SYBR PCR mix (Fermentas, K0222);1 μL 10 μM forward primer;1 μL 10 μM reverse primer;x μL ChIP-ed DNA (input control, or genomic DNA);y μL nuclease-free H2 O to adjust the reaction volume to 20 μL. .. For each primer pair, set up qPCR in duplicate in a 96-well plate with genomic DNA from step 3a.1, input control and ChIP-ed DNA:10 μL 2x SYBR PCR mix (Fermentas, K0222);1 μL 10 μM forward primer;1 μL 10 μM reverse primer;x μL ChIP-ed DNA (input control, or genomic DNA);y μL nuclease-free H2 O to adjust the reaction volume to 20 μL.

    Article Title: A Bacterial Artificial Chromosome Transgene with Polymorphic Cd72 Inhibits the Development of Glomerulonephritis and Vasculitis in MRL-Faslpr Lupus Mice
    Article Snippet: The following primers in the RP23-195K8 BAC sequences were used for the transgene 5′-GAACACGCATTGGCCTACTC-3′ (forward), 5′-TCATCGGGAAGGTAGCCAATC-3′ (reverse) and the reference gene ( mIL2 ), 5′-CTAGGCCACAGAATTGAAAGATCT-3′ (forward), 5′-GTAGGTGGAAATTCTAGCATCATCC-3′ (reverse). .. The Platinum SYBR Green qPCR SuperMix UDG (Invitrogen) was used for the real-time amplification, according to the manufacturer’s protocol. .. The assays were performed in duplicate on an ABI PRISM 7700 system (Applied Biosystems).

    Article Title: Overexpression of DNA methyltransferase 1 as a negative independent prognostic factor in primary gastrointestinal diffuse large B-cell lymphoma treated with CHOP-like regimen and rituximab
    Article Snippet: Following this, reverse transcription reactions were performed using a PrimeScript 1st Strand cDNA Synthesis Kit (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's instructions and qPCR was performed using a CM9600 Sequence Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). .. The amplification step was performed in a total volume of 20 µl, with 10 µl kit-supplied QuantiTect™ SYBR® Green RT-PCR Master mix (Applied Biosystems Life Technologies, Foster City, CA, USA), 0.4 µl of each primer (10 µM), 2 µl of cDNA (50 ng RNA) and 7.2 µl ddH2 O. .. The PCR cycling parameters were set as follows: 95°C for 30 sec, followed by 40 cycles of PCR reacting at 95°C for 5 sec, and finally 60°C for 34 sec. β actin was used as an internal standard.

    Filtration:

    Article Title: Addressing the Instability of DNA Nanostructures in Tissue Culture
    Article Snippet: SYBR Safe stain and CyQuant direct cell proliferation assay were purchased from Life Technologies Corporation. .. Carbon Formvar grids and uranyl formate were purchased from Electron Microscopy Sciences.

    Positive Control:

    Article Title: Trypanosome co-infections increase in a declining marsupial population
    Article Snippet: Four controls were used in both internal and external PCR reactions; a pre- and post-PCR negative control, a negative control using dH2 O as a PCR template, and a PCR positive control, using either T. copemani or T. vegrandis DNA. .. PCR products were run on a 2% agarose gel using SYBR Safe Gel Stain (Invitrogen, California USA) and visualized by illumination with LED light.

    Synthesized:

    Article Title: Neuronal TRPV1 activation regulates alveolar bone resorption by suppressing osteoclastogenesis via CGRP
    Article Snippet: Total RNA was isolated from tissues and cells using TRI Reagent® (Molecular Research Center, Inc., Cincinnati, OH, USA). cDNA was synthesized using Transcriptor Universal cDNA Master (Roche Molecular Systems, Inc., Branchburg, NJ, USA). .. PCR products were run on 1.5% agarose gels and visualized using SYBR® Safe DNA (Invitrogen Corporation, Carlsbad, CA, USA).

    Lambda DNA Preparation:

    Article Title: RalR (a DNase) and RalA (a small RNA) form a type I toxin-antitoxin system in Escherichia coli
    Article Snippet: Purified RalR and RalR-mutant (150 pmol each) were incubated separately with different substrates at 37°C in 250 mM NaCl, 100 mM Tris-HCl (pH 7.5), 10 mM CaCl2 and 10 mM MgCl2 for 30 min and 120 min. Plasmid pBR322 isolated from BW25113, genomic DNA of BW25113, as well as methylated and unmethylated lambda DNA (dam − , dcm − ) (1 μg) were used as substrates, respectively. .. The reaction was stopped by the addition of a stop solution (25% glycerol, 0.5% SDS, 0.05% bromophenol blue and 50 mM EDTA) and was analyzed by electrophoresis on 1% agarose gels stained with SYBR safe (Invitrogen).

    Quantitative RT-PCR:

    Article Title: Chromatin conformation regulates the coordination between DNA replication and transcription
    Article Snippet: One microgram of RNA per time point was treated with DNAse (Roche), and reverse transcription reactions were performed with SuperScript III First Strand Synthesis System (Invitrogen) using random hexamers. .. Pre-mRNAs at the different time-points were quantified by RT-qPCR with primers spanning different exon–intron junctions (Supplementary Table ). qPCR reactions were performed with an ABI Prism 7900HT Detection System (Applied Biosystems), with HotStar Taq polymerase (Qiagen) and SYBR (Molecular Probes). qPCR conditions were empirically tested for each primer pair and only those showing and slope of −3.3 +-10% and R2 value > 0.99 were accepted. .. Quantitative analyses were carried out using the ABI Prism 7900HT SDS Software (v 2.4).

    Article Title: CagA increases DNA methylation and decreases PTEN expression in human gastric cancer
    Article Snippet: Paragraph title: Reverse transcription (RT)-qPCR ... PCR was performed using a SYBR qPCR kit (Invitrogen; Thermo Fisher Scientific, Inc.).

    SYBR Green Assay:

    Article Title: A Bacterial Artificial Chromosome Transgene with Polymorphic Cd72 Inhibits the Development of Glomerulonephritis and Vasculitis in MRL-Faslpr Lupus Mice
    Article Snippet: The following primers in the RP23-195K8 BAC sequences were used for the transgene 5′-GAACACGCATTGGCCTACTC-3′ (forward), 5′-TCATCGGGAAGGTAGCCAATC-3′ (reverse) and the reference gene ( mIL2 ), 5′-CTAGGCCACAGAATTGAAAGATCT-3′ (forward), 5′-GTAGGTGGAAATTCTAGCATCATCC-3′ (reverse). .. The Platinum SYBR Green qPCR SuperMix UDG (Invitrogen) was used for the real-time amplification, according to the manufacturer’s protocol. .. The assays were performed in duplicate on an ABI PRISM 7700 system (Applied Biosystems).

    Article Title: Overexpression of DNA methyltransferase 1 as a negative independent prognostic factor in primary gastrointestinal diffuse large B-cell lymphoma treated with CHOP-like regimen and rituximab
    Article Snippet: Following this, reverse transcription reactions were performed using a PrimeScript 1st Strand cDNA Synthesis Kit (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's instructions and qPCR was performed using a CM9600 Sequence Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). .. The amplification step was performed in a total volume of 20 µl, with 10 µl kit-supplied QuantiTect™ SYBR® Green RT-PCR Master mix (Applied Biosystems Life Technologies, Foster City, CA, USA), 0.4 µl of each primer (10 µM), 2 µl of cDNA (50 ng RNA) and 7.2 µl ddH2 O. .. The PCR cycling parameters were set as follows: 95°C for 30 sec, followed by 40 cycles of PCR reacting at 95°C for 5 sec, and finally 60°C for 34 sec. β actin was used as an internal standard.

    Incubation:

    Article Title: RalR (a DNase) and RalA (a small RNA) form a type I toxin-antitoxin system in Escherichia coli
    Article Snippet: Purified RalR and RalR-mutant (150 pmol each) were incubated separately with different substrates at 37°C in 250 mM NaCl, 100 mM Tris-HCl (pH 7.5), 10 mM CaCl2 and 10 mM MgCl2 for 30 min and 120 min. Plasmid pBR322 isolated from BW25113, genomic DNA of BW25113, as well as methylated and unmethylated lambda DNA (dam − , dcm − ) (1 μg) were used as substrates, respectively. .. The reaction was stopped by the addition of a stop solution (25% glycerol, 0.5% SDS, 0.05% bromophenol blue and 50 mM EDTA) and was analyzed by electrophoresis on 1% agarose gels stained with SYBR safe (Invitrogen).

    Article Title: Chromatin conformation regulates the coordination between DNA replication and transcription
    Article Snippet: Briefly, subconfluent cell cultures were incubated in complete medium supplemented with 100 μM DRB for 3 h, then DRB-medium was washed-off and fresh medium was added to resume transcription elongation. .. Pre-mRNAs at the different time-points were quantified by RT-qPCR with primers spanning different exon–intron junctions (Supplementary Table ). qPCR reactions were performed with an ABI Prism 7900HT Detection System (Applied Biosystems), with HotStar Taq polymerase (Qiagen) and SYBR (Molecular Probes). qPCR conditions were empirically tested for each primer pair and only those showing and slope of −3.3 +-10% and R2 value > 0.99 were accepted.

    Article Title: RecA-dependent programmable endonuclease Ref cleaves DNA in two distinct steps
    Article Snippet: ATP or ATPγS (3 mM) was added and incubated for additional 20 min, followed by M13mp18 cdsDNA (8 µMnt) and another 20-min incubation. .. Samples were analyzed by electrophoresis on a 0.8% agarose gel with Tris-acetate + ethylenediaminetetraacetic acid buffer, stained with SYBR Gold nucleic acid gel stain (Invitrogen) and imaged using the SYBR gold settings on a Typhoon variable mode imager (GE Healthcare).

    Proliferation Assay:

    Article Title: Addressing the Instability of DNA Nanostructures in Tissue Culture
    Article Snippet: Accugene 10× TBE buffer, PCR tubes, and 96-well PCR plates (Axygen) were purchased from VWR. .. SYBR Safe stain and CyQuant direct cell proliferation assay were purchased from Life Technologies Corporation. .. Glycerol, Tris base, EDTA, Triton X-114, Tween20, magnesium chloride, magnesium sulfate, and sodium chloride were purchased from Sigma-Aldrich.

    Cell Culture:

    Article Title: Raman Microspectroscopy Analysis in the Treatment of Acanthamoeba Keratitis
    Article Snippet: Paragraph title: Cell Culture Protocol ... Bands were visualised on SYBR SafeTM DNA gel stain (Invitrogen Corporation, Italy) stained 1% agarose gels.

    Expressing:

    Article Title: CagA increases DNA methylation and decreases PTEN expression in human gastric cancer
    Article Snippet: PCR was performed using a SYBR qPCR kit (Invitrogen; Thermo Fisher Scientific, Inc.). .. The PCR conditions were as follows: Predenaturation at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 30 sec and polymerization at 70°C for 45 sec, followed by 1 cycle of melting curve at 95°C for 15 sec, 60°C for 60 sec and 95°C for 15 sec. PCR was performed using a CFX96 Touch™ Real-time PCR Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

    Article Title: DNA damage as a consequence of NLR activation
    Article Snippet: Total RNA was extracted using TRI reagent (Sigma) and performed according to the manufacturer’s recommendations. .. 1μg of total RNA was used for DNase treatment with TURBO DNA-free Kit (Ambion Life technologies). cDNA synthesis was then performed using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher) according to the manufacturer’s recommendations. qPCR was done using the Luminaris SYBR ROX qPCR Master Mix (ThermoFisher) and expression level was normalized to UBQ10 (primers listed in ). .. We thank Gitta Coaker for the Arabidopsis line harboring Dex- inducible avrRpm1 .

    Modification:

    Article Title: Human papillomavirus, high-grade intraepithelial neoplasia and killer immunoglogulin-like receptors: a Western Australian cohort study
    Article Snippet: Genomic DNA was extracted from cytology specimens by a modified EDNA HiSpEx™ tissue kit (Saturn Biotech, Perth, Australia) method in which ThinPrep cervical samples were mixed by shaking and inversion, a 500 μl aliquot was removed and mixed with 1 mL of PCR grade water in a microfuge tube. .. PCR products were electrophoresed on a 2% agarose gel in 1× TAE buffer precast in SYBR Safe (Invitrogen, Carlsbad, CA) and photographed under a UV transilluminator.

    Article Title: Chitosan/hyaluronic acid/plasmid-DNA nanoparticles encoding interleukin-1 receptor antagonist attenuate inflammation in synoviocytes induced by interleukin-1 beta
    Article Snippet: Deoxyribonuclease I (DNase I), SYBR Prime Ex Taq II were obtained from Invitrogen (USA). .. Deoxyribonuclease I (DNase I), SYBR Prime Ex Taq II were obtained from Invitrogen (USA).

    Real-time Polymerase Chain Reaction:

    Article Title: Serological and molecular epidemiology of canine adenovirus type 1 in red foxes (Vulpes vulpes) in the United Kingdom
    Article Snippet: The sensitivities of the PCRs for detecting CAV-1 and CAV-2 were assessed using dilutions of plasmid controls containing a CAV-1 or CAV-2 insert (see ‘Quantitative PCR’) and was determined to be less than 10 copies. .. PCR products were loaded on agarose gels stained with SYBR safe DNA stain (Invitrogen, Paisley, UK) and separated by electrophoresis.

    Article Title: Chromatin conformation regulates the coordination between DNA replication and transcription
    Article Snippet: One microgram of RNA per time point was treated with DNAse (Roche), and reverse transcription reactions were performed with SuperScript III First Strand Synthesis System (Invitrogen) using random hexamers. .. Pre-mRNAs at the different time-points were quantified by RT-qPCR with primers spanning different exon–intron junctions (Supplementary Table ). qPCR reactions were performed with an ABI Prism 7900HT Detection System (Applied Biosystems), with HotStar Taq polymerase (Qiagen) and SYBR (Molecular Probes). qPCR conditions were empirically tested for each primer pair and only those showing and slope of −3.3 +-10% and R2 value > 0.99 were accepted. .. Quantitative analyses were carried out using the ABI Prism 7900HT SDS Software (v 2.4).

    Article Title: Locus-specific ChIP combined with NGS analysis reveals genomic regulatory regions that physically interact with the Pax5 promoter in a chicken B cell line
    Article Snippet: ChIP assays were performed with DT40 cells (2 × 106 ) and each antibody (3.5 µl for H3K4me1 or 2 µg for the others) as described previously. .. DNA purified using ChIP DNA Clean & Concentrator (Zymo Research) was used as template for real-time PCR with SYBR Select Master Mix (Applied Biosystems) on Applied Biosystems 7900HT Fast Real-Time PCR System. .. The scheme of iChIP-Seq used in this study is as follows ( ): (I) Using homologous recombination, binding elements of the bacterial DNA-binding protein LexA (LexA BE) were inserted ∼0.3 kb upstream from the transcription start site (TSS) of the Pax5 exon 1A in chromosome Z of DT40 cells. (II) 3xFNLDD, which consists of 3xFLAG-tag, a nuclear localization signal (NLS), and LexA DNA-binding and dimerization domains, was expressed in the cells established in Step (I). (III) The resultant cells were crosslinked with formaldehyde and lysed, and chromatin DNA was fragmented by sonication. (IV) The tagged locus (the Pax5 promoter region) was affinity-purified using an anti-FLAG antibody. (V) After reverse crosslinking and DNA purification, genomic regions interacting with the Pax5 promoter region were identified by NGS analysis.

    Article Title: CagA increases DNA methylation and decreases PTEN expression in human gastric cancer
    Article Snippet: The specific primers used for RT-qPCR are listed in . .. PCR was performed using a SYBR qPCR kit (Invitrogen; Thermo Fisher Scientific, Inc.). .. The PCR conditions were as follows: Predenaturation at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 30 sec and polymerization at 70°C for 45 sec, followed by 1 cycle of melting curve at 95°C for 15 sec, 60°C for 60 sec and 95°C for 15 sec. PCR was performed using a CFX96 Touch™ Real-time PCR Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

    Article Title: ANRIL Promoter DNA Methylation: A Perinatal Marker for Later Adiposity
    Article Snippet: Methylation ranges are shown in Table S13 and primers in Table S14. .. 2.12 RNA was extracted from whole umbilical cords obtained in the GUSTO study, using the RNeasy Mini Kit (Qiagen) as per manufacturer's guidelines. cDNA was prepared using the Hi-Capacity cDNA Reverse Transcription Kit (Life Technologies), and qPCR reactions run in triplicate with Power SYBR (Life Technologies) on a ABI 7900HT as per manufacturer's guidelines. .. Primer sequences are shown in Table S14 Primers to detect circular transcripts are described in .

    Article Title: DNA damage as a consequence of NLR activation
    Article Snippet: Total RNA was extracted using TRI reagent (Sigma) and performed according to the manufacturer’s recommendations. .. 1μg of total RNA was used for DNase treatment with TURBO DNA-free Kit (Ambion Life technologies). cDNA synthesis was then performed using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher) according to the manufacturer’s recommendations. qPCR was done using the Luminaris SYBR ROX qPCR Master Mix (ThermoFisher) and expression level was normalized to UBQ10 (primers listed in ). .. We thank Gitta Coaker for the Arabidopsis line harboring Dex- inducible avrRpm1 .

    Article Title: Chromatin Immunoprecipitation (ChIP) using Drosophila tissue
    Article Snippet: Dilute genomic DNA at the following concentration to make a standard curve: undiluted, 1/10, 1/100, 1/1000, 1/5000. .. For each primer pair, set up qPCR in duplicate in a 96-well plate with genomic DNA from step 3a.1, input control and ChIP-ed DNA:10 μL 2x SYBR PCR mix (Fermentas, K0222);1 μL 10 μM forward primer;1 μL 10 μM reverse primer;x μL ChIP-ed DNA (input control, or genomic DNA);y μL nuclease-free H2 O to adjust the reaction volume to 20 μL. .. Spin down the 96-well plate with Mini plate spinner (Labnet).

    Article Title: Effect of circadian clock mutations on DNA damage response in mammalian cells
    Article Snippet: Total RNA from mouse liver and mouse skin fibroblasts, isolated using TRIZOL reagent (Invitrogen), was subjected to reverse transcription using ImProm-II Reverse Transcription System (Promega). .. Real-time PCR experiments were performed using an ABI 7300 system (Applied Biosystems) and MaximaTM SYBR Green/ROX qPCR Master Mix (Fermentas). .. RT-PCR cycle thresholds of individual genes were normalized to the corresponding actin mRNA expression values obtained with the primer set.

    Article Title: A Bacterial Artificial Chromosome Transgene with Polymorphic Cd72 Inhibits the Development of Glomerulonephritis and Vasculitis in MRL-Faslpr Lupus Mice
    Article Snippet: The following primers in the RP23-195K8 BAC sequences were used for the transgene 5′-GAACACGCATTGGCCTACTC-3′ (forward), 5′-TCATCGGGAAGGTAGCCAATC-3′ (reverse) and the reference gene ( mIL2 ), 5′-CTAGGCCACAGAATTGAAAGATCT-3′ (forward), 5′-GTAGGTGGAAATTCTAGCATCATCC-3′ (reverse). .. The Platinum SYBR Green qPCR SuperMix UDG (Invitrogen) was used for the real-time amplification, according to the manufacturer’s protocol. .. The assays were performed in duplicate on an ABI PRISM 7700 system (Applied Biosystems).

    Article Title: Overexpression of DNA methyltransferase 1 as a negative independent prognostic factor in primary gastrointestinal diffuse large B-cell lymphoma treated with CHOP-like regimen and rituximab
    Article Snippet: Paragraph title: RNA, cDNA preparation and qPCR ... The amplification step was performed in a total volume of 20 µl, with 10 µl kit-supplied QuantiTect™ SYBR® Green RT-PCR Master mix (Applied Biosystems Life Technologies, Foster City, CA, USA), 0.4 µl of each primer (10 µM), 2 µl of cDNA (50 ng RNA) and 7.2 µl ddH2 O.

    Electron Microscopy:

    Article Title: Addressing the Instability of DNA Nanostructures in Tissue Culture
    Article Snippet: SYBR Safe stain and CyQuant direct cell proliferation assay were purchased from Life Technologies Corporation. .. SYBR Safe stain and CyQuant direct cell proliferation assay were purchased from Life Technologies Corporation.

    Low Copy Number:

    Article Title: Serological and molecular epidemiology of canine adenovirus type 1 in red foxes (Vulpes vulpes) in the United Kingdom
    Article Snippet: Each DNA sample in this study was tested in triplicate, in an attempt to increase rates of detection of very low copy number samples and to negate possible false positive results from contamination. .. PCR products were loaded on agarose gels stained with SYBR safe DNA stain (Invitrogen, Paisley, UK) and separated by electrophoresis.

    Infection:

    Article Title: Human papillomavirus, high-grade intraepithelial neoplasia and killer immunoglogulin-like receptors: a Western Australian cohort study
    Article Snippet: It is recognized that cells containing integrated HPV would have genomic alterations; however the majority of cells within the ThinPrep sample would not be infected with HPV and would have normal genomes, and so were considered suitable for KIR typing by PCR. .. PCR products were electrophoresed on a 2% agarose gel in 1× TAE buffer precast in SYBR Safe (Invitrogen, Carlsbad, CA) and photographed under a UV transilluminator.

    CyQUANT Assay:

    Article Title: Addressing the Instability of DNA Nanostructures in Tissue Culture
    Article Snippet: Accugene 10× TBE buffer, PCR tubes, and 96-well PCR plates (Axygen) were purchased from VWR. .. SYBR Safe stain and CyQuant direct cell proliferation assay were purchased from Life Technologies Corporation. .. Glycerol, Tris base, EDTA, Triton X-114, Tween20, magnesium chloride, magnesium sulfate, and sodium chloride were purchased from Sigma-Aldrich.

    Inhibition:

    Article Title: Chromatin conformation regulates the coordination between DNA replication and transcription
    Article Snippet: Transient inhibition of RNAPII Ser2 phosphorylation with DRB and release was adapted from Singh and Padget (2009) following the modifications of Jimeno-González et al. . .. Pre-mRNAs at the different time-points were quantified by RT-qPCR with primers spanning different exon–intron junctions (Supplementary Table ). qPCR reactions were performed with an ABI Prism 7900HT Detection System (Applied Biosystems), with HotStar Taq polymerase (Qiagen) and SYBR (Molecular Probes). qPCR conditions were empirically tested for each primer pair and only those showing and slope of −3.3 +-10% and R2 value > 0.99 were accepted.

    Sequencing:

    Article Title: Serological and molecular epidemiology of canine adenovirus type 1 in red foxes (Vulpes vulpes) in the United Kingdom
    Article Snippet: PCR products were loaded on agarose gels stained with SYBR safe DNA stain (Invitrogen, Paisley, UK) and separated by electrophoresis. .. The expected amplicon size of the second round product was 188 base pairs (bp) for both CAV-1 and CAV-2 primer sets.

    Article Title: Raman Microspectroscopy Analysis in the Treatment of Acanthamoeba Keratitis
    Article Snippet: Bands were visualised on SYBR SafeTM DNA gel stain (Invitrogen Corporation, Italy) stained 1% agarose gels. .. PCR products were purified using Nucleo Spin Extract II (Macherey-Nagel GmbH & Co. KG ) purification kit and sequenced for both strands by the Bio-Fab Research (Italy).

    Article Title: Overexpression of DNA methyltransferase 1 as a negative independent prognostic factor in primary gastrointestinal diffuse large B-cell lymphoma treated with CHOP-like regimen and rituximab
    Article Snippet: Following this, reverse transcription reactions were performed using a PrimeScript 1st Strand cDNA Synthesis Kit (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's instructions and qPCR was performed using a CM9600 Sequence Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). .. The amplification step was performed in a total volume of 20 µl, with 10 µl kit-supplied QuantiTect™ SYBR® Green RT-PCR Master mix (Applied Biosystems Life Technologies, Foster City, CA, USA), 0.4 µl of each primer (10 µM), 2 µl of cDNA (50 ng RNA) and 7.2 µl ddH2 O.

    Molecular Weight:

    Article Title: Chitosan/hyaluronic acid/plasmid-DNA nanoparticles encoding interleukin-1 receptor antagonist attenuate inflammation in synoviocytes induced by interleukin-1 beta
    Article Snippet: Sodium hyaluronate (molecular weight, 35 kDa) was purchased from Freda Biochem Co., Ltd. (Shandong, China). .. Deoxyribonuclease I (DNase I), SYBR Prime Ex Taq II were obtained from Invitrogen (USA).

    DNA Extraction:

    Article Title: Human papillomavirus, high-grade intraepithelial neoplasia and killer immunoglogulin-like receptors: a Western Australian cohort study
    Article Snippet: Paragraph title: DNA extraction and PCR ... PCR products were electrophoresed on a 2% agarose gel in 1× TAE buffer precast in SYBR Safe (Invitrogen, Carlsbad, CA) and photographed under a UV transilluminator.

    Article Title: Trypanosome co-infections increase in a declining marsupial population
    Article Snippet: Paragraph title: DNA extraction and Trypanosoma spp. detection by PCR ... PCR products were run on a 2% agarose gel using SYBR Safe Gel Stain (Invitrogen, California USA) and visualized by illumination with LED light.

    Article Title: Raman Microspectroscopy Analysis in the Treatment of Acanthamoeba Keratitis
    Article Snippet: A 405-bp region of the 18S-rRNA gene (ASA.S1) that includes the Diagnostic Fragment 3 (DF3) was amplified using the genus-specific primers JDP1 and JDP2 to a final volume of 25 µ l. The PCR program was as follows: denaturing step at 96°C for 2 min, followed by 35 cycles of denaturing for 1 min at 96°C, annealing for 1 min at 60°C and extension for 1 min at 72°C, followed by a final extension at 72°C for 7 min. Sterile distilled water was included as negative control in each batch of DNA extraction and PCR reactions. .. Bands were visualised on SYBR SafeTM DNA gel stain (Invitrogen Corporation, Italy) stained 1% agarose gels.

    Nucleic Acid Electrophoresis:

    Article Title: Neuronal TRPV1 activation regulates alveolar bone resorption by suppressing osteoclastogenesis via CGRP
    Article Snippet: Paragraph title: Polymerase chain reaction (PCR) and gel electrophoresis ... PCR products were run on 1.5% agarose gels and visualized using SYBR® Safe DNA (Invitrogen Corporation, Carlsbad, CA, USA).

    Article Title: Quantitative methodology is critical for assessing DNA methylation and impacts on correlation with patient outcome
    Article Snippet: Paragraph title: Gel electrophoresis analysis ... Four microliters of the SMART-MSP product were resolved on a 3.5% agarose gel stained with SYBR Safe (Invitrogen), at 80 mV over 1.5 hours.

    DNA Cleavage Assay:

    Article Title: RalR (a DNase) and RalA (a small RNA) form a type I toxin-antitoxin system in Escherichia coli
    Article Snippet: Paragraph title: DNA cleavage assay ... The reaction was stopped by the addition of a stop solution (25% glycerol, 0.5% SDS, 0.05% bromophenol blue and 50 mM EDTA) and was analyzed by electrophoresis on 1% agarose gels stained with SYBR safe (Invitrogen).

    Methylation:

    Article Title: RalR (a DNase) and RalA (a small RNA) form a type I toxin-antitoxin system in Escherichia coli
    Article Snippet: Purified RalR and RalR-mutant (150 pmol each) were incubated separately with different substrates at 37°C in 250 mM NaCl, 100 mM Tris-HCl (pH 7.5), 10 mM CaCl2 and 10 mM MgCl2 for 30 min and 120 min. Plasmid pBR322 isolated from BW25113, genomic DNA of BW25113, as well as methylated and unmethylated lambda DNA (dam − , dcm − ) (1 μg) were used as substrates, respectively. .. The reaction was stopped by the addition of a stop solution (25% glycerol, 0.5% SDS, 0.05% bromophenol blue and 50 mM EDTA) and was analyzed by electrophoresis on 1% agarose gels stained with SYBR safe (Invitrogen).

    Article Title: CagA increases DNA methylation and decreases PTEN expression in human gastric cancer
    Article Snippet: All these genes have been reported to have close associations with gene methylation/demethylation ( ). .. PCR was performed using a SYBR qPCR kit (Invitrogen; Thermo Fisher Scientific, Inc.).

    Isolation:

    Article Title: Neuronal TRPV1 activation regulates alveolar bone resorption by suppressing osteoclastogenesis via CGRP
    Article Snippet: Total RNA was isolated from tissues and cells using TRI Reagent® (Molecular Research Center, Inc., Cincinnati, OH, USA). cDNA was synthesized using Transcriptor Universal cDNA Master (Roche Molecular Systems, Inc., Branchburg, NJ, USA). .. PCR products were run on 1.5% agarose gels and visualized using SYBR® Safe DNA (Invitrogen Corporation, Carlsbad, CA, USA).

    Article Title: RalR (a DNase) and RalA (a small RNA) form a type I toxin-antitoxin system in Escherichia coli
    Article Snippet: Purified RalR and RalR-mutant (150 pmol each) were incubated separately with different substrates at 37°C in 250 mM NaCl, 100 mM Tris-HCl (pH 7.5), 10 mM CaCl2 and 10 mM MgCl2 for 30 min and 120 min. Plasmid pBR322 isolated from BW25113, genomic DNA of BW25113, as well as methylated and unmethylated lambda DNA (dam − , dcm − ) (1 μg) were used as substrates, respectively. .. The reaction was stopped by the addition of a stop solution (25% glycerol, 0.5% SDS, 0.05% bromophenol blue and 50 mM EDTA) and was analyzed by electrophoresis on 1% agarose gels stained with SYBR safe (Invitrogen).

    Article Title: Chromatin conformation regulates the coordination between DNA replication and transcription
    Article Snippet: Total RNA from equivalent number of cells was isolated every 10 min with RNeasy kit (Quiagen) following the manufacturer´s instructions. .. Pre-mRNAs at the different time-points were quantified by RT-qPCR with primers spanning different exon–intron junctions (Supplementary Table ). qPCR reactions were performed with an ABI Prism 7900HT Detection System (Applied Biosystems), with HotStar Taq polymerase (Qiagen) and SYBR (Molecular Probes). qPCR conditions were empirically tested for each primer pair and only those showing and slope of −3.3 +-10% and R2 value > 0.99 were accepted.

    Article Title: Raman Microspectroscopy Analysis in the Treatment of Acanthamoeba Keratitis
    Article Snippet: The sediment was suspended and isolation of Acanthamoeba was performed on 1,5% non nutrient agar (Difco, Milan, Italy) enriched by Escherichia coli K12. .. Bands were visualised on SYBR SafeTM DNA gel stain (Invitrogen Corporation, Italy) stained 1% agarose gels.

    Article Title: Effect of circadian clock mutations on DNA damage response in mammalian cells
    Article Snippet: Paragraph title: RNA isolation and real-time PCR ... Real-time PCR experiments were performed using an ABI 7300 system (Applied Biosystems) and MaximaTM SYBR Green/ROX qPCR Master Mix (Fermentas).

    Polymerase Chain Reaction:

    Article Title: Neuronal TRPV1 activation regulates alveolar bone resorption by suppressing osteoclastogenesis via CGRP
    Article Snippet: Conventional PCR was performed in a 20 μL reaction volume using GoTaq polymerase (Promega Corporation, Madison, WI, USA) with the following protocol: predenaturation at 94 °C for 5 min followed by 30 cycles of denaturation at 94 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 30 s, and a final extension step at 72 °C for 10 min by using a GeneAmp® PCR System 7700 (Applied Biosystems, Carlsbad, CA, USA). .. PCR products were run on 1.5% agarose gels and visualized using SYBR® Safe DNA (Invitrogen Corporation, Carlsbad, CA, USA). .. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh ) was amplified using forward primer 5′-TCAACAGCAACTCCCACTCTT-3′ and reverse primer 5′-ACCCTGTTGCTGTAGCCGTAT-3′.

    Article Title: Human papillomavirus, high-grade intraepithelial neoplasia and killer immunoglogulin-like receptors: a Western Australian cohort study
    Article Snippet: In each typing run 10th International Histocompatibility Workshop cell lines, CB6B and JBUSH, were used as positive controls [ ]. .. PCR products were electrophoresed on a 2% agarose gel in 1× TAE buffer precast in SYBR Safe (Invitrogen, Carlsbad, CA) and photographed under a UV transilluminator. .. KIR gene frequencies in high grade CIN cases and controls, and in HPV 16/18 and non 16/18 derived high grade CIN, were compared by χ 2 analysis with Yates’ correction.

    Article Title: Serological and molecular epidemiology of canine adenovirus type 1 in red foxes (Vulpes vulpes) in the United Kingdom
    Article Snippet: Each DNA sample in this study was tested in triplicate, in an attempt to increase rates of detection of very low copy number samples and to negate possible false positive results from contamination. .. PCR products were loaded on agarose gels stained with SYBR safe DNA stain (Invitrogen, Paisley, UK) and separated by electrophoresis. .. Amplicons were observed using a G:Box gel viewing system (Syngene, Cambridge, UK).

    Article Title: Trypanosome co-infections increase in a declining marsupial population
    Article Snippet: A negative control from the DNA extraction was also run in each PCR. .. PCR products were run on a 2% agarose gel using SYBR Safe Gel Stain (Invitrogen, California USA) and visualized by illumination with LED light. .. 2.3 First, to understand how the prevalence of each parasite varied in context of the population decline of the host, we estimated the prevalence (using Jeffrey's confidence intervals) of infection among individuals in each sampling trip, and graphed this against time, and the capture rates (percentage of traps occupied in a session) of woylies for each sampling trip.

    Article Title: Structure, organization and tissue expression of the pig SLC13A1 and SLC13A4 sulfate transporter genes
    Article Snippet: Each PCR included 200 nM forward and reverse primers, first strand cDNA (equivalent to 0.2 µg RNA), 1.6 mM dNTPs and 1 Unit DNA polymerase (Scientifix). .. The thermal cycling protocol was: 35 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min; followed by 72 °C for 5 min. PCR-amplified DNA was size-fractioned in a 1.5% agarose gel and then visualised using SYBR® safe DNA stain (Invitrogen) under UV light. .. 3 In this study, we report the pig SLC13A1 and SLC13A4 gene, cDNA, 5’-flanking region and protein sequences, as well as the tissue distribution of SLC13A1 and SLC13A4 mRNA, and compare those to the previously published findings for the human and mouse homologues.

    Article Title: Addressing the Instability of DNA Nanostructures in Tissue Culture
    Article Snippet: Accugene 10× TBE buffer, PCR tubes, and 96-well PCR plates (Axygen) were purchased from VWR. .. SYBR Safe stain and CyQuant direct cell proliferation assay were purchased from Life Technologies Corporation.

    Article Title: Raman Microspectroscopy Analysis in the Treatment of Acanthamoeba Keratitis
    Article Snippet: A 405-bp region of the 18S-rRNA gene (ASA.S1) that includes the Diagnostic Fragment 3 (DF3) was amplified using the genus-specific primers JDP1 and JDP2 to a final volume of 25 µ l. The PCR program was as follows: denaturing step at 96°C for 2 min, followed by 35 cycles of denaturing for 1 min at 96°C, annealing for 1 min at 60°C and extension for 1 min at 72°C, followed by a final extension at 72°C for 7 min. Sterile distilled water was included as negative control in each batch of DNA extraction and PCR reactions. .. Bands were visualised on SYBR SafeTM DNA gel stain (Invitrogen Corporation, Italy) stained 1% agarose gels.

    Article Title: CagA increases DNA methylation and decreases PTEN expression in human gastric cancer
    Article Snippet: The specific primers used for RT-qPCR are listed in . .. PCR was performed using a SYBR qPCR kit (Invitrogen; Thermo Fisher Scientific, Inc.). .. The PCR conditions were as follows: Predenaturation at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 30 sec and polymerization at 70°C for 45 sec, followed by 1 cycle of melting curve at 95°C for 15 sec, 60°C for 60 sec and 95°C for 15 sec. PCR was performed using a CFX96 Touch™ Real-time PCR Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

    Article Title: Chromatin Immunoprecipitation (ChIP) using Drosophila tissue
    Article Snippet: Dilute genomic DNA at the following concentration to make a standard curve: undiluted, 1/10, 1/100, 1/1000, 1/5000. .. For each primer pair, set up qPCR in duplicate in a 96-well plate with genomic DNA from step 3a.1, input control and ChIP-ed DNA:10 μL 2x SYBR PCR mix (Fermentas, K0222);1 μL 10 μM forward primer;1 μL 10 μM reverse primer;x μL ChIP-ed DNA (input control, or genomic DNA);y μL nuclease-free H2 O to adjust the reaction volume to 20 μL. .. Spin down the 96-well plate with Mini plate spinner (Labnet).

    Article Title: Effect of circadian clock mutations on DNA damage response in mammalian cells
    Article Snippet: Real-time PCR experiments were performed using an ABI 7300 system (Applied Biosystems) and MaximaTM SYBR Green/ROX qPCR Master Mix (Fermentas). .. RT-PCR cycle thresholds of individual genes were normalized to the corresponding actin mRNA expression values obtained with the primer set.

    Article Title: A Bacterial Artificial Chromosome Transgene with Polymorphic Cd72 Inhibits the Development of Glomerulonephritis and Vasculitis in MRL-Faslpr Lupus Mice
    Article Snippet: The Platinum SYBR Green qPCR SuperMix UDG (Invitrogen) was used for the real-time amplification, according to the manufacturer’s protocol. .. The Platinum SYBR Green qPCR SuperMix UDG (Invitrogen) was used for the real-time amplification, according to the manufacturer’s protocol.

    Negative Control:

    Article Title: RalR (a DNase) and RalA (a small RNA) form a type I toxin-antitoxin system in Escherichia coli
    Article Snippet: The reaction was stopped by the addition of a stop solution (25% glycerol, 0.5% SDS, 0.05% bromophenol blue and 50 mM EDTA) and was analyzed by electrophoresis on 1% agarose gels stained with SYBR safe (Invitrogen). .. To study whether Ca2+ and Mg2+ are cofactors for RalR, 250 mM NaCl and 100 mM Tris-HCl (pH 7.5) buffer with or without the addition of 10 mM Ca2+ or 10 mM Mg2+ were used to conduct the DNA cleavage assay.

    Article Title: Trypanosome co-infections increase in a declining marsupial population
    Article Snippet: PCR products were run on a 2% agarose gel using SYBR Safe Gel Stain (Invitrogen, California USA) and visualized by illumination with LED light. .. PCR products were run on a 2% agarose gel using SYBR Safe Gel Stain (Invitrogen, California USA) and visualized by illumination with LED light.

    Article Title: Raman Microspectroscopy Analysis in the Treatment of Acanthamoeba Keratitis
    Article Snippet: A 405-bp region of the 18S-rRNA gene (ASA.S1) that includes the Diagnostic Fragment 3 (DF3) was amplified using the genus-specific primers JDP1 and JDP2 to a final volume of 25 µ l. The PCR program was as follows: denaturing step at 96°C for 2 min, followed by 35 cycles of denaturing for 1 min at 96°C, annealing for 1 min at 60°C and extension for 1 min at 72°C, followed by a final extension at 72°C for 7 min. Sterile distilled water was included as negative control in each batch of DNA extraction and PCR reactions. .. Bands were visualised on SYBR SafeTM DNA gel stain (Invitrogen Corporation, Italy) stained 1% agarose gels.

    Purification:

    Article Title: RalR (a DNase) and RalA (a small RNA) form a type I toxin-antitoxin system in Escherichia coli
    Article Snippet: Purified RalR and RalR-mutant (150 pmol each) were incubated separately with different substrates at 37°C in 250 mM NaCl, 100 mM Tris-HCl (pH 7.5), 10 mM CaCl2 and 10 mM MgCl2 for 30 min and 120 min. Plasmid pBR322 isolated from BW25113, genomic DNA of BW25113, as well as methylated and unmethylated lambda DNA (dam − , dcm − ) (1 μg) were used as substrates, respectively. .. The reaction was stopped by the addition of a stop solution (25% glycerol, 0.5% SDS, 0.05% bromophenol blue and 50 mM EDTA) and was analyzed by electrophoresis on 1% agarose gels stained with SYBR safe (Invitrogen).

    Article Title: Locus-specific ChIP combined with NGS analysis reveals genomic regulatory regions that physically interact with the Pax5 promoter in a chicken B cell line
    Article Snippet: ChIP assays were performed with DT40 cells (2 × 106 ) and each antibody (3.5 µl for H3K4me1 or 2 µg for the others) as described previously. .. DNA purified using ChIP DNA Clean & Concentrator (Zymo Research) was used as template for real-time PCR with SYBR Select Master Mix (Applied Biosystems) on Applied Biosystems 7900HT Fast Real-Time PCR System. .. The scheme of iChIP-Seq used in this study is as follows ( ): (I) Using homologous recombination, binding elements of the bacterial DNA-binding protein LexA (LexA BE) were inserted ∼0.3 kb upstream from the transcription start site (TSS) of the Pax5 exon 1A in chromosome Z of DT40 cells. (II) 3xFNLDD, which consists of 3xFLAG-tag, a nuclear localization signal (NLS), and LexA DNA-binding and dimerization domains, was expressed in the cells established in Step (I). (III) The resultant cells were crosslinked with formaldehyde and lysed, and chromatin DNA was fragmented by sonication. (IV) The tagged locus (the Pax5 promoter region) was affinity-purified using an anti-FLAG antibody. (V) After reverse crosslinking and DNA purification, genomic regions interacting with the Pax5 promoter region were identified by NGS analysis.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Overexpression of DNA methyltransferase 1 as a negative independent prognostic factor in primary gastrointestinal diffuse large B-cell lymphoma treated with CHOP-like regimen and rituximab
    Article Snippet: Following this, reverse transcription reactions were performed using a PrimeScript 1st Strand cDNA Synthesis Kit (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's instructions and qPCR was performed using a CM9600 Sequence Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). .. The amplification step was performed in a total volume of 20 µl, with 10 µl kit-supplied QuantiTect™ SYBR® Green RT-PCR Master mix (Applied Biosystems Life Technologies, Foster City, CA, USA), 0.4 µl of each primer (10 µM), 2 µl of cDNA (50 ng RNA) and 7.2 µl ddH2 O. .. The PCR cycling parameters were set as follows: 95°C for 30 sec, followed by 40 cycles of PCR reacting at 95°C for 5 sec, and finally 60°C for 34 sec. β actin was used as an internal standard.

    Nested PCR:

    Article Title: Trypanosome co-infections increase in a declining marsupial population
    Article Snippet: A general Trypanosoma PCR was carried out first to screen samples , and for samples that were positive, two separate clade-specific nested PCR protocols were used to amplify the trypanosome 18S rDNA region. .. PCR products were run on a 2% agarose gel using SYBR Safe Gel Stain (Invitrogen, California USA) and visualized by illumination with LED light.

    Mouse Assay:

    Article Title: A Bacterial Artificial Chromosome Transgene with Polymorphic Cd72 Inhibits the Development of Glomerulonephritis and Vasculitis in MRL-Faslpr Lupus Mice
    Article Snippet: The Platinum SYBR Green qPCR SuperMix UDG (Invitrogen) was used for the real-time amplification, according to the manufacturer’s protocol. .. The Platinum SYBR Green qPCR SuperMix UDG (Invitrogen) was used for the real-time amplification, according to the manufacturer’s protocol.

    Chromatin Immunoprecipitation:

    Article Title: Locus-specific ChIP combined with NGS analysis reveals genomic regulatory regions that physically interact with the Pax5 promoter in a chicken B cell line
    Article Snippet: ChIP assays were performed with DT40 cells (2 × 106 ) and each antibody (3.5 µl for H3K4me1 or 2 µg for the others) as described previously. .. DNA purified using ChIP DNA Clean & Concentrator (Zymo Research) was used as template for real-time PCR with SYBR Select Master Mix (Applied Biosystems) on Applied Biosystems 7900HT Fast Real-Time PCR System. .. The scheme of iChIP-Seq used in this study is as follows ( ): (I) Using homologous recombination, binding elements of the bacterial DNA-binding protein LexA (LexA BE) were inserted ∼0.3 kb upstream from the transcription start site (TSS) of the Pax5 exon 1A in chromosome Z of DT40 cells. (II) 3xFNLDD, which consists of 3xFLAG-tag, a nuclear localization signal (NLS), and LexA DNA-binding and dimerization domains, was expressed in the cells established in Step (I). (III) The resultant cells were crosslinked with formaldehyde and lysed, and chromatin DNA was fragmented by sonication. (IV) The tagged locus (the Pax5 promoter region) was affinity-purified using an anti-FLAG antibody. (V) After reverse crosslinking and DNA purification, genomic regions interacting with the Pax5 promoter region were identified by NGS analysis.

    Article Title: Chromatin Immunoprecipitation (ChIP) using Drosophila tissue
    Article Snippet: Dilute genomic DNA at the following concentration to make a standard curve: undiluted, 1/10, 1/100, 1/1000, 1/5000. .. For each primer pair, set up qPCR in duplicate in a 96-well plate with genomic DNA from step 3a.1, input control and ChIP-ed DNA:10 μL 2x SYBR PCR mix (Fermentas, K0222);1 μL 10 μM forward primer;1 μL 10 μM reverse primer;x μL ChIP-ed DNA (input control, or genomic DNA);y μL nuclease-free H2 O to adjust the reaction volume to 20 μL. .. Spin down the 96-well plate with Mini plate spinner (Labnet).

    Plasmid Preparation:

    Article Title: RalR (a DNase) and RalA (a small RNA) form a type I toxin-antitoxin system in Escherichia coli
    Article Snippet: Purified RalR and RalR-mutant (150 pmol each) were incubated separately with different substrates at 37°C in 250 mM NaCl, 100 mM Tris-HCl (pH 7.5), 10 mM CaCl2 and 10 mM MgCl2 for 30 min and 120 min. Plasmid pBR322 isolated from BW25113, genomic DNA of BW25113, as well as methylated and unmethylated lambda DNA (dam − , dcm − ) (1 μg) were used as substrates, respectively. .. The reaction was stopped by the addition of a stop solution (25% glycerol, 0.5% SDS, 0.05% bromophenol blue and 50 mM EDTA) and was analyzed by electrophoresis on 1% agarose gels stained with SYBR safe (Invitrogen).

    Article Title: Serological and molecular epidemiology of canine adenovirus type 1 in red foxes (Vulpes vulpes) in the United Kingdom
    Article Snippet: The sensitivities of the PCRs for detecting CAV-1 and CAV-2 were assessed using dilutions of plasmid controls containing a CAV-1 or CAV-2 insert (see ‘Quantitative PCR’) and was determined to be less than 10 copies. .. PCR products were loaded on agarose gels stained with SYBR safe DNA stain (Invitrogen, Paisley, UK) and separated by electrophoresis.

    Article Title: A Bacterial Artificial Chromosome Transgene with Polymorphic Cd72 Inhibits the Development of Glomerulonephritis and Vasculitis in MRL-Faslpr Lupus Mice
    Article Snippet: The Platinum SYBR Green qPCR SuperMix UDG (Invitrogen) was used for the real-time amplification, according to the manufacturer’s protocol. .. The Platinum SYBR Green qPCR SuperMix UDG (Invitrogen) was used for the real-time amplification, according to the manufacturer’s protocol.

    Electrophoresis:

    Article Title: RalR (a DNase) and RalA (a small RNA) form a type I toxin-antitoxin system in Escherichia coli
    Article Snippet: Purified RalR and RalR-mutant (150 pmol each) were incubated separately with different substrates at 37°C in 250 mM NaCl, 100 mM Tris-HCl (pH 7.5), 10 mM CaCl2 and 10 mM MgCl2 for 30 min and 120 min. Plasmid pBR322 isolated from BW25113, genomic DNA of BW25113, as well as methylated and unmethylated lambda DNA (dam − , dcm − ) (1 μg) were used as substrates, respectively. .. The reaction was stopped by the addition of a stop solution (25% glycerol, 0.5% SDS, 0.05% bromophenol blue and 50 mM EDTA) and was analyzed by electrophoresis on 1% agarose gels stained with SYBR safe (Invitrogen). .. The equal amount of deoxyribonuclease I (DNase I; New England Biolabs, Beverly, MA, USA) was used as a positive control, and excess EDTA (20 mM) was added in the reaction to inhibit the nuclease activity.

    Article Title: Serological and molecular epidemiology of canine adenovirus type 1 in red foxes (Vulpes vulpes) in the United Kingdom
    Article Snippet: Each DNA sample in this study was tested in triplicate, in an attempt to increase rates of detection of very low copy number samples and to negate possible false positive results from contamination. .. PCR products were loaded on agarose gels stained with SYBR safe DNA stain (Invitrogen, Paisley, UK) and separated by electrophoresis. .. Amplicons were observed using a G:Box gel viewing system (Syngene, Cambridge, UK).

    RNA Extraction:

    Article Title: DNA damage as a consequence of NLR activation
    Article Snippet: Paragraph title: RNA extraction & quantitative real-time PCR (qPCR) ... 1μg of total RNA was used for DNase treatment with TURBO DNA-free Kit (Ambion Life technologies). cDNA synthesis was then performed using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher) according to the manufacturer’s recommendations. qPCR was done using the Luminaris SYBR ROX qPCR Master Mix (ThermoFisher) and expression level was normalized to UBQ10 (primers listed in ).

    Article Title: Overexpression of DNA methyltransferase 1 as a negative independent prognostic factor in primary gastrointestinal diffuse large B-cell lymphoma treated with CHOP-like regimen and rituximab
    Article Snippet: For the RNA extraction step, ∼25 mg of tumor tissue was pulverized under liquid nitrogen using a pestle and mortar. .. The amplification step was performed in a total volume of 20 µl, with 10 µl kit-supplied QuantiTect™ SYBR® Green RT-PCR Master mix (Applied Biosystems Life Technologies, Foster City, CA, USA), 0.4 µl of each primer (10 µM), 2 µl of cDNA (50 ng RNA) and 7.2 µl ddH2 O.

    Agarose Gel Electrophoresis:

    Article Title: Human papillomavirus, high-grade intraepithelial neoplasia and killer immunoglogulin-like receptors: a Western Australian cohort study
    Article Snippet: In each typing run 10th International Histocompatibility Workshop cell lines, CB6B and JBUSH, were used as positive controls [ ]. .. PCR products were electrophoresed on a 2% agarose gel in 1× TAE buffer precast in SYBR Safe (Invitrogen, Carlsbad, CA) and photographed under a UV transilluminator. .. KIR gene frequencies in high grade CIN cases and controls, and in HPV 16/18 and non 16/18 derived high grade CIN, were compared by χ 2 analysis with Yates’ correction.

    Article Title: Quantitative methodology is critical for assessing DNA methylation and impacts on correlation with patient outcome
    Article Snippet: The raw run data for COL2A1, DAPK1 and RASSF1A were generated by the Rotor-Gene Q (Qiagen) and exported into LinRegPCR (version 2012.3.0.0) for the estimation of amplification efficiency, after accounting for bias introduced by variable baseline fluorescence [ ]. .. Four microliters of the SMART-MSP product were resolved on a 3.5% agarose gel stained with SYBR Safe (Invitrogen), at 80 mV over 1.5 hours. .. The preliminary statistical analysis of the MS-HRM results indicated a significant correlation with worse outcome for samples demonstrating mostly heterogeneous methylation of RUNX3 and ABO .

    Article Title: Trypanosome co-infections increase in a declining marsupial population
    Article Snippet: A negative control from the DNA extraction was also run in each PCR. .. PCR products were run on a 2% agarose gel using SYBR Safe Gel Stain (Invitrogen, California USA) and visualized by illumination with LED light. .. 2.3 First, to understand how the prevalence of each parasite varied in context of the population decline of the host, we estimated the prevalence (using Jeffrey's confidence intervals) of infection among individuals in each sampling trip, and graphed this against time, and the capture rates (percentage of traps occupied in a session) of woylies for each sampling trip.

    Article Title: Structure, organization and tissue expression of the pig SLC13A1 and SLC13A4 sulfate transporter genes
    Article Snippet: Each PCR included 200 nM forward and reverse primers, first strand cDNA (equivalent to 0.2 µg RNA), 1.6 mM dNTPs and 1 Unit DNA polymerase (Scientifix). .. The thermal cycling protocol was: 35 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min; followed by 72 °C for 5 min. PCR-amplified DNA was size-fractioned in a 1.5% agarose gel and then visualised using SYBR® safe DNA stain (Invitrogen) under UV light. .. 3 In this study, we report the pig SLC13A1 and SLC13A4 gene, cDNA, 5’-flanking region and protein sequences, as well as the tissue distribution of SLC13A1 and SLC13A4 mRNA, and compare those to the previously published findings for the human and mouse homologues.

    Article Title: RecA-dependent programmable endonuclease Ref cleaves DNA in two distinct steps
    Article Snippet: The reactions were stopped at the noted time points by removing 20 µL from the reaction and adding it to 20 µl of stop solution [12 mM Tris-acetate, pH 7.5, 10.8% (w/v) Ficoll, 0.15% (w/v) each bromophenol blue and xylene cyanol, 8% sodium dodecyl sulphate] and incubating at 37°C for additional 30 min. .. Samples were analyzed by electrophoresis on a 0.8% agarose gel with Tris-acetate + ethylenediaminetetraacetic acid buffer, stained with SYBR Gold nucleic acid gel stain (Invitrogen) and imaged using the SYBR gold settings on a Typhoon variable mode imager (GE Healthcare). .. Gel image was analyzed using ImageQuantTL software (GE Healthcare).

    Concentration Assay:

    Article Title: Chromatin Immunoprecipitation (ChIP) using Drosophila tissue
    Article Snippet: Dilute genomic DNA at the following concentration to make a standard curve: undiluted, 1/10, 1/100, 1/1000, 1/5000. .. For each primer pair, set up qPCR in duplicate in a 96-well plate with genomic DNA from step 3a.1, input control and ChIP-ed DNA:10 μL 2x SYBR PCR mix (Fermentas, K0222);1 μL 10 μM forward primer;1 μL 10 μM reverse primer;x μL ChIP-ed DNA (input control, or genomic DNA);y μL nuclease-free H2 O to adjust the reaction volume to 20 μL.

    BAC Assay:

    Article Title: A Bacterial Artificial Chromosome Transgene with Polymorphic Cd72 Inhibits the Development of Glomerulonephritis and Vasculitis in MRL-Faslpr Lupus Mice
    Article Snippet: The following primers in the RP23-195K8 BAC sequences were used for the transgene 5′-GAACACGCATTGGCCTACTC-3′ (forward), 5′-TCATCGGGAAGGTAGCCAATC-3′ (reverse) and the reference gene ( mIL2 ), 5′-CTAGGCCACAGAATTGAAAGATCT-3′ (forward), 5′-GTAGGTGGAAATTCTAGCATCATCC-3′ (reverse). .. The Platinum SYBR Green qPCR SuperMix UDG (Invitrogen) was used for the real-time amplification, according to the manufacturer’s protocol.

    Staining:

    Article Title: RalR (a DNase) and RalA (a small RNA) form a type I toxin-antitoxin system in Escherichia coli
    Article Snippet: Purified RalR and RalR-mutant (150 pmol each) were incubated separately with different substrates at 37°C in 250 mM NaCl, 100 mM Tris-HCl (pH 7.5), 10 mM CaCl2 and 10 mM MgCl2 for 30 min and 120 min. Plasmid pBR322 isolated from BW25113, genomic DNA of BW25113, as well as methylated and unmethylated lambda DNA (dam − , dcm − ) (1 μg) were used as substrates, respectively. .. The reaction was stopped by the addition of a stop solution (25% glycerol, 0.5% SDS, 0.05% bromophenol blue and 50 mM EDTA) and was analyzed by electrophoresis on 1% agarose gels stained with SYBR safe (Invitrogen). .. The equal amount of deoxyribonuclease I (DNase I; New England Biolabs, Beverly, MA, USA) was used as a positive control, and excess EDTA (20 mM) was added in the reaction to inhibit the nuclease activity.

    Article Title: Quantitative methodology is critical for assessing DNA methylation and impacts on correlation with patient outcome
    Article Snippet: The raw run data for COL2A1, DAPK1 and RASSF1A were generated by the Rotor-Gene Q (Qiagen) and exported into LinRegPCR (version 2012.3.0.0) for the estimation of amplification efficiency, after accounting for bias introduced by variable baseline fluorescence [ ]. .. Four microliters of the SMART-MSP product were resolved on a 3.5% agarose gel stained with SYBR Safe (Invitrogen), at 80 mV over 1.5 hours. .. The preliminary statistical analysis of the MS-HRM results indicated a significant correlation with worse outcome for samples demonstrating mostly heterogeneous methylation of RUNX3 and ABO .

    Article Title: Serological and molecular epidemiology of canine adenovirus type 1 in red foxes (Vulpes vulpes) in the United Kingdom
    Article Snippet: Each DNA sample in this study was tested in triplicate, in an attempt to increase rates of detection of very low copy number samples and to negate possible false positive results from contamination. .. PCR products were loaded on agarose gels stained with SYBR safe DNA stain (Invitrogen, Paisley, UK) and separated by electrophoresis. .. Amplicons were observed using a G:Box gel viewing system (Syngene, Cambridge, UK).

    Article Title: Trypanosome co-infections increase in a declining marsupial population
    Article Snippet: A negative control from the DNA extraction was also run in each PCR. .. PCR products were run on a 2% agarose gel using SYBR Safe Gel Stain (Invitrogen, California USA) and visualized by illumination with LED light. .. 2.3 First, to understand how the prevalence of each parasite varied in context of the population decline of the host, we estimated the prevalence (using Jeffrey's confidence intervals) of infection among individuals in each sampling trip, and graphed this against time, and the capture rates (percentage of traps occupied in a session) of woylies for each sampling trip.

    Article Title: Structure, organization and tissue expression of the pig SLC13A1 and SLC13A4 sulfate transporter genes
    Article Snippet: Each PCR included 200 nM forward and reverse primers, first strand cDNA (equivalent to 0.2 µg RNA), 1.6 mM dNTPs and 1 Unit DNA polymerase (Scientifix). .. The thermal cycling protocol was: 35 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min; followed by 72 °C for 5 min. PCR-amplified DNA was size-fractioned in a 1.5% agarose gel and then visualised using SYBR® safe DNA stain (Invitrogen) under UV light. .. 3 In this study, we report the pig SLC13A1 and SLC13A4 gene, cDNA, 5’-flanking region and protein sequences, as well as the tissue distribution of SLC13A1 and SLC13A4 mRNA, and compare those to the previously published findings for the human and mouse homologues.

    Article Title: Addressing the Instability of DNA Nanostructures in Tissue Culture
    Article Snippet: Accugene 10× TBE buffer, PCR tubes, and 96-well PCR plates (Axygen) were purchased from VWR. .. SYBR Safe stain and CyQuant direct cell proliferation assay were purchased from Life Technologies Corporation. .. Glycerol, Tris base, EDTA, Triton X-114, Tween20, magnesium chloride, magnesium sulfate, and sodium chloride were purchased from Sigma-Aldrich.

    Article Title: Raman Microspectroscopy Analysis in the Treatment of Acanthamoeba Keratitis
    Article Snippet: A 405-bp region of the 18S-rRNA gene (ASA.S1) that includes the Diagnostic Fragment 3 (DF3) was amplified using the genus-specific primers JDP1 and JDP2 to a final volume of 25 µ l. The PCR program was as follows: denaturing step at 96°C for 2 min, followed by 35 cycles of denaturing for 1 min at 96°C, annealing for 1 min at 60°C and extension for 1 min at 72°C, followed by a final extension at 72°C for 7 min. Sterile distilled water was included as negative control in each batch of DNA extraction and PCR reactions. .. Bands were visualised on SYBR SafeTM DNA gel stain (Invitrogen Corporation, Italy) stained 1% agarose gels. .. PCR products were purified using Nucleo Spin Extract II (Macherey-Nagel GmbH & Co. KG ) purification kit and sequenced for both strands by the Bio-Fab Research (Italy).

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    Thermo Fisher sybr select master mix
    Sybr Select Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 503 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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