Structured Review

Promega dntps
Single-cell transcriptome analysis uncovers distinct proliferative populations within the murine heart. ( A ) Experimental timeline for tissue collection of hearts from wild-type and Mki67 RFP mice, either neonatal or adults, 14 d after sham, ischemia/reperfusion (I/R), or MI surgery ( n = 2–4 mice per condition). ( B ) Schematic representation of <t>SORT-seq</t> workflow. Hearts were isolated (1) and digested into single-cell suspension (2), and Ki67-RFP + and Ki67-RFP − cells were sorted into 384-well plates containing primers, <t>dNTPs,</t> and spike-ins (3). Retrotranscription mix was distributed using Nanodrop II, and material was pooled and amplified (4) before pair-end sequencing (5). Cells were clustered using RaceID2 (6). ( C ) Clustering of cardiac cells and cell-to-cell distances visualized by t -distributed stochastic neighbor-embedding ( t -SNE) map, highlighting identified major cardiac cell types. ( D ) Numbers of cells assigned to each cardiac cell lineage. ( E ) t -SNE map highlighting identified cell types based on previously described cellular markers (logarithmic scale of transcript expression). Markers expression is shown in Lower panel by immunofluorescent staining. (Scale bars: 50 μm.) ( F ) t -SNE map displaying cell cycle stage of each cell [S (red), G 2 /M (green), G 0 /G 1 (blue)] assigned by the cyclone algorithm. ( G ) t -SNE map showing the Ki67-RFP status from the flow cytometry data; Ki67-RFP + (red), Ki67-RFP − (black), or Mki67 wt/wt cells without TagRFP construct (gray) and radar plot showing Ki67-RFP + cells enriched for the cycling G 2 /M stage according to the cyclone algorithm. Asterisks indicate significance (χ 2 test: *** P
Dntps, supplied by Promega, used in various techniques. Bioz Stars score: 97/100, based on 169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/dntps/product/Promega
Average 97 stars, based on 169 article reviews
Price from $9.99 to $1999.99
dntps - by Bioz Stars, 2020-01
97/100 stars

Images

1) Product Images from "Profiling proliferative cells and their progeny in damaged murine hearts"

Article Title: Profiling proliferative cells and their progeny in damaged murine hearts

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1805829115

Single-cell transcriptome analysis uncovers distinct proliferative populations within the murine heart. ( A ) Experimental timeline for tissue collection of hearts from wild-type and Mki67 RFP mice, either neonatal or adults, 14 d after sham, ischemia/reperfusion (I/R), or MI surgery ( n = 2–4 mice per condition). ( B ) Schematic representation of SORT-seq workflow. Hearts were isolated (1) and digested into single-cell suspension (2), and Ki67-RFP + and Ki67-RFP − cells were sorted into 384-well plates containing primers, dNTPs, and spike-ins (3). Retrotranscription mix was distributed using Nanodrop II, and material was pooled and amplified (4) before pair-end sequencing (5). Cells were clustered using RaceID2 (6). ( C ) Clustering of cardiac cells and cell-to-cell distances visualized by t -distributed stochastic neighbor-embedding ( t -SNE) map, highlighting identified major cardiac cell types. ( D ) Numbers of cells assigned to each cardiac cell lineage. ( E ) t -SNE map highlighting identified cell types based on previously described cellular markers (logarithmic scale of transcript expression). Markers expression is shown in Lower panel by immunofluorescent staining. (Scale bars: 50 μm.) ( F ) t -SNE map displaying cell cycle stage of each cell [S (red), G 2 /M (green), G 0 /G 1 (blue)] assigned by the cyclone algorithm. ( G ) t -SNE map showing the Ki67-RFP status from the flow cytometry data; Ki67-RFP + (red), Ki67-RFP − (black), or Mki67 wt/wt cells without TagRFP construct (gray) and radar plot showing Ki67-RFP + cells enriched for the cycling G 2 /M stage according to the cyclone algorithm. Asterisks indicate significance (χ 2 test: *** P
Figure Legend Snippet: Single-cell transcriptome analysis uncovers distinct proliferative populations within the murine heart. ( A ) Experimental timeline for tissue collection of hearts from wild-type and Mki67 RFP mice, either neonatal or adults, 14 d after sham, ischemia/reperfusion (I/R), or MI surgery ( n = 2–4 mice per condition). ( B ) Schematic representation of SORT-seq workflow. Hearts were isolated (1) and digested into single-cell suspension (2), and Ki67-RFP + and Ki67-RFP − cells were sorted into 384-well plates containing primers, dNTPs, and spike-ins (3). Retrotranscription mix was distributed using Nanodrop II, and material was pooled and amplified (4) before pair-end sequencing (5). Cells were clustered using RaceID2 (6). ( C ) Clustering of cardiac cells and cell-to-cell distances visualized by t -distributed stochastic neighbor-embedding ( t -SNE) map, highlighting identified major cardiac cell types. ( D ) Numbers of cells assigned to each cardiac cell lineage. ( E ) t -SNE map highlighting identified cell types based on previously described cellular markers (logarithmic scale of transcript expression). Markers expression is shown in Lower panel by immunofluorescent staining. (Scale bars: 50 μm.) ( F ) t -SNE map displaying cell cycle stage of each cell [S (red), G 2 /M (green), G 0 /G 1 (blue)] assigned by the cyclone algorithm. ( G ) t -SNE map showing the Ki67-RFP status from the flow cytometry data; Ki67-RFP + (red), Ki67-RFP − (black), or Mki67 wt/wt cells without TagRFP construct (gray) and radar plot showing Ki67-RFP + cells enriched for the cycling G 2 /M stage according to the cyclone algorithm. Asterisks indicate significance (χ 2 test: *** P

Techniques Used: Mouse Assay, Isolation, Amplification, Sequencing, Expressing, Staining, Flow Cytometry, Cytometry, Construct

2) Product Images from "Profiling proliferative cells and their progeny in damaged murine hearts"

Article Title: Profiling proliferative cells and their progeny in damaged murine hearts

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1805829115

Single-cell transcriptome analysis uncovers distinct proliferative populations within the murine heart. ( A ) Experimental timeline for tissue collection of hearts from wild-type and Mki67 RFP mice, either neonatal or adults, 14 d after sham, ischemia/reperfusion (I/R), or MI surgery ( n = 2–4 mice per condition). ( B ) Schematic representation of SORT-seq workflow. Hearts were isolated (1) and digested into single-cell suspension (2), and Ki67-RFP + and Ki67-RFP − cells were sorted into 384-well plates containing primers, dNTPs, and spike-ins (3). Retrotranscription mix was distributed using Nanodrop II, and material was pooled and amplified (4) before pair-end sequencing (5). Cells were clustered using RaceID2 (6). ( C ) Clustering of cardiac cells and cell-to-cell distances visualized by t -distributed stochastic neighbor-embedding ( t -SNE) map, highlighting identified major cardiac cell types. ( D ) Numbers of cells assigned to each cardiac cell lineage. ( E ) t -SNE map highlighting identified cell types based on previously described cellular markers (logarithmic scale of transcript expression). Markers expression is shown in Lower panel by immunofluorescent staining. (Scale bars: 50 μm.) ( F ) t -SNE map displaying cell cycle stage of each cell [S (red), G 2 /M (green), G 0 /G 1 (blue)] assigned by the cyclone algorithm. ( G ) t -SNE map showing the Ki67-RFP status from the flow cytometry data; Ki67-RFP + (red), Ki67-RFP − (black), or Mki67 wt/wt cells without TagRFP construct (gray) and radar plot showing Ki67-RFP + cells enriched for the cycling G 2 /M stage according to the cyclone algorithm. Asterisks indicate significance (χ 2 test: *** P
Figure Legend Snippet: Single-cell transcriptome analysis uncovers distinct proliferative populations within the murine heart. ( A ) Experimental timeline for tissue collection of hearts from wild-type and Mki67 RFP mice, either neonatal or adults, 14 d after sham, ischemia/reperfusion (I/R), or MI surgery ( n = 2–4 mice per condition). ( B ) Schematic representation of SORT-seq workflow. Hearts were isolated (1) and digested into single-cell suspension (2), and Ki67-RFP + and Ki67-RFP − cells were sorted into 384-well plates containing primers, dNTPs, and spike-ins (3). Retrotranscription mix was distributed using Nanodrop II, and material was pooled and amplified (4) before pair-end sequencing (5). Cells were clustered using RaceID2 (6). ( C ) Clustering of cardiac cells and cell-to-cell distances visualized by t -distributed stochastic neighbor-embedding ( t -SNE) map, highlighting identified major cardiac cell types. ( D ) Numbers of cells assigned to each cardiac cell lineage. ( E ) t -SNE map highlighting identified cell types based on previously described cellular markers (logarithmic scale of transcript expression). Markers expression is shown in Lower panel by immunofluorescent staining. (Scale bars: 50 μm.) ( F ) t -SNE map displaying cell cycle stage of each cell [S (red), G 2 /M (green), G 0 /G 1 (blue)] assigned by the cyclone algorithm. ( G ) t -SNE map showing the Ki67-RFP status from the flow cytometry data; Ki67-RFP + (red), Ki67-RFP − (black), or Mki67 wt/wt cells without TagRFP construct (gray) and radar plot showing Ki67-RFP + cells enriched for the cycling G 2 /M stage according to the cyclone algorithm. Asterisks indicate significance (χ 2 test: *** P

Techniques Used: Mouse Assay, Isolation, Amplification, Sequencing, Expressing, Staining, Flow Cytometry, Cytometry, Construct

Related Articles

Clone Assay:

Article Title: Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer
Article Snippet: The mixture was then used in a 20 μL reverse transcription reaction containing 200 μmol•L¯¹ each dNTPs and 20 U of reverse transcriptase (Promega), which was incubated at 37 °C for 1 h. The RNA-cDNA mixture (5 μL) was then used in 50 μL PCR reaction mixture containing all four dNTPs at 60 μmol•L¯¹, 5 U of Taq polymerase (Promega), and 50 pmol•L¯¹ of appropriate 5’ and 3’ primers[ , ]. .. VK1a and VK3a are 5’ primers for amplification of the κ chain with the Sac I site for cloning into the vector pComb3.

Article Title: The Staphylococcus aureus superantigen SElX is a bifunctional toxin that inhibits neutrophil functionStaphylococcal enterotoxin-like X (SElX) is a unique superantigen with functional features of two major families of staphylococcal virulence factors
Article Snippet: Primers were used at a final concentration of 250 nM along with 2 mM dNTPs (Promega, Hampshire, UK). .. Following digestion, 1 μl of the amplified vector product was transformed into E . coli Solopack cells from the Strataclone blunt cloning kit (Agilent technologies, UK) following the manufacturer’s instructions.

Amplification:

Article Title: CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes
Article Snippet: Paragraph title: Laser capture microdissection, RNA amplification, and reverse transcriptase-PCR (RT-PCR) ... A 50 μL PCR reaction was run with the following final concentrations: 450 ng of each primer (see for PCR primer sequences), 2 mM MgCl2 , 0.3 mM dNTPs, 2.5 U Taq polymerase (Promega GoFlex DNA polymerase) and 1 μL of 10 ng/μL DNA.

Article Title: Pre-treatment or Post-treatment of Human Glioma Cells With BIX01294, the Inhibitor of Histone Methyltransferase G9a, Sensitizes Cells to Temozolomide
Article Snippet: The modified DNA was then amplified using primers specific for either methylated or unmethylated MGMT gene promoter sequences, as listed in Supplementary Table . .. Each PCR mixture contained 1 μl of DNA, 500 nM of primers, 1x reaction buffer containing 1.5 mM MgCl2, 1 U HotStarTaq DNA Polymerase and 250 mM dNTPs (Promega, United States).

Article Title: The First Identified Citrus tristeza virus Isolate of Turkey Contains a Mixture of Mild and Severe Strains
Article Snippet: Paragraph title: Amplification of the CP and RdRp genes by RT-PCR ... First, 5 ml of dsRNA was denatured at 95ºC for 5 min and quickly chilled on ice. cDNA was synthesized from denatured dsRNA in 20 μl mixture containing 1X AMV first strand buffer (50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 ), 20 units RNAsin (Promega, USA), 0.5 mM dNTPs, 20 units AMV reverse transcriptase (Promega, USA) and 20 pmol random hexamers using the MJ Mini thermal cycler PTC1148 (Bio-Rad, USA) programmed at 30ºC for 10 min. followed by 42ºC for 60 min and 75ºC for 15 min. PCR was conducted in 50 μl reaction mixture containing 1X Pfu reaction buffer [20 mM Tris-HCl (pH 8.8 at 25°C), 10 mM (NH4 )2 SO4 , 100 mM KCl, 1% (v/v) Triton X-100, 1 mg/ml BSA and 20 mM MgSO4 ], 0.2 mM dNTPs, 2.5 unit Pfu DNA polymerase (Promega, USA) 5 μl of cDNA and 20 pmol of primer specific to CP (BC24 and 25) and RdRp (BC42 and BC43) genes.

Article Title: Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer
Article Snippet: The mixture was then used in a 20 μL reverse transcription reaction containing 200 μmol•L¯¹ each dNTPs and 20 U of reverse transcriptase (Promega), which was incubated at 37 °C for 1 h. The RNA-cDNA mixture (5 μL) was then used in 50 μL PCR reaction mixture containing all four dNTPs at 60 μmol•L¯¹, 5 U of Taq polymerase (Promega), and 50 pmol•L¯¹ of appropriate 5’ and 3’ primers[ , ]. .. VK1a and VK3a are 5’ primers for amplification of the κ chain with the Sac I site for cloning into the vector pComb3.

Article Title: The Staphylococcus aureus superantigen SElX is a bifunctional toxin that inhibits neutrophil functionStaphylococcal enterotoxin-like X (SElX) is a unique superantigen with functional features of two major families of staphylococcal virulence factors
Article Snippet: Primers were used at a final concentration of 250 nM along with 2 mM dNTPs (Promega, Hampshire, UK). .. Following digestion, 1 μl of the amplified vector product was transformed into E . coli Solopack cells from the Strataclone blunt cloning kit (Agilent technologies, UK) following the manufacturer’s instructions.

Article Title: Homeostatic regulation of T cell trafficking by a B cell derived peptide is impaired in autoimmune and chronic inflammatory disease
Article Snippet: To convert mRNA to cDNA, random primers (Promega, Maddison, USA) were annealed to 1 μg of mRNA for 5 minutes at 70°C, after which the following mastermix was added to give a final volume of 30 μl: 10 U Superscript II Reverse Transcriptase (RT), 10 U RNAout RNase inhibitor, 1X Superscript Buffer (all from Invitrogen) and 10 mM dNTPs (Promega). .. Samples were amplified in duplicates using the 7500HT Real-Time PCR machine (Applied Biosystems) and analyzed using the software package SDS 2.2 (Applied Biosystems).

Expressing:

Article Title: Homeostatic regulation of T cell trafficking by a B cell derived peptide is impaired in autoimmune and chronic inflammatory disease
Article Snippet: To convert mRNA to cDNA, random primers (Promega, Maddison, USA) were annealed to 1 μg of mRNA for 5 minutes at 70°C, after which the following mastermix was added to give a final volume of 30 μl: 10 U Superscript II Reverse Transcriptase (RT), 10 U RNAout RNase inhibitor, 1X Superscript Buffer (all from Invitrogen) and 10 mM dNTPs (Promega). .. Data were expressed as relative expression units relative to 18S or as fold change (2^−deltadelta Ct method).

Synthesized:

Article Title: The First Identified Citrus tristeza virus Isolate of Turkey Contains a Mixture of Mild and Severe Strains
Article Snippet: .. First, 5 ml of dsRNA was denatured at 95ºC for 5 min and quickly chilled on ice. cDNA was synthesized from denatured dsRNA in 20 μl mixture containing 1X AMV first strand buffer (50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 ), 20 units RNAsin (Promega, USA), 0.5 mM dNTPs, 20 units AMV reverse transcriptase (Promega, USA) and 20 pmol random hexamers using the MJ Mini thermal cycler PTC1148 (Bio-Rad, USA) programmed at 30ºC for 10 min. followed by 42ºC for 60 min and 75ºC for 15 min. PCR was conducted in 50 μl reaction mixture containing 1X Pfu reaction buffer [20 mM Tris-HCl (pH 8.8 at 25°C), 10 mM (NH4 )2 SO4 , 100 mM KCl, 1% (v/v) Triton X-100, 1 mg/ml BSA and 20 mM MgSO4 ], 0.2 mM dNTPs, 2.5 unit Pfu DNA polymerase (Promega, USA) 5 μl of cDNA and 20 pmol of primer specific to CP (BC24 and 25) and RdRp (BC42 and BC43) genes. .. PCR was performed in the MJ Mini thermal cycler PTC1148 (Bio-Rad, USA) programmed for initial denaturation at 94°C for 3 min and then, 40 cycles of denaturation at 94°C for 30 s, primer annealing at 55°C for 30 s, and primer extension at 72°C for 1 min for the CP gene and 2 min for the RdRp gene followed by final extension at 72°C for 5 min. PCR products were separated in 1% agarose gel with 100 bp DNA size marker, stained with ethidium bromide and visualized and analyzed by Doc-It system (UVP, England).

Article Title: Chronic hyperoxia alters the expression of neurotrophic factors in the carotid body of neonatal rats
Article Snippet: .. For BDNF, GDNF, and Ret, first strand cDNA was synthesized from 150 ng total RNA using 200U Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA), 1 μl primer mix (200 ng/μl oligo dT’s and 50 ng/μl random hexamers) (Promega, Madison, WI, USA), 1 μl 10 mM dNTPs (Promega), 4 μl 5× First strand buffer (Invitrogen), 2 μl 0.1 M dithiothreitol (DTT) (Invitrogen), and 1 μl RNAseOUT (Invitrogen); these reactions were incubated at 25°C for 5 min followed by 42°C for 50 min and 70°C for 15 min. For TrkB, Cbln1, and Fgf2, cDNA was synthesized from 200 ng total RNA using 1 μl ImProm-II Reverse Transcriptase (Promega), 1 μl primer mix (200 ng/μl oligo dT’s and 50 ng/μl random hexamers) (Promega), 1 μl 10 mM dNTPs (Promega), 4 μl ImProm-II 5× First strand buffer (Promega), 2 μl 25 mM MgCl2 (Promega), and 1 μl RNAseOUT (Invitrogen); these reactions were incubated at 25°C for 5 min followed by 42°C for 60 min and 70°C for 15 min. For Vgf, cDNA was synthesized from 300 ng total RNA using RT2 First Strand Kit (SABiosciences, Frederick, MD USA) according to kit instructions with the exception of replacing GE Buffer with water. .. The resulting single-stranded cDNA products were stored at −20°C until quantitative PCR analysis.

Neutralization:

Article Title: Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications
Article Snippet: GsI-IIC RT (400 nM) was preincubated with the annealed R2/R2R-G heteroduplex (50 nM) and 50-nt acceptor template RNA (100 nM) in 10 μl of reaction medium containing 200 mM NaCl, 5 mM MgCl2 , 20 mM Tris-HCl pH 7.5, and 5 mM DTT for 30 min at room temperature, prior to initiating template-switching reverse transcription reactions by adding 0.4 μL of 25 mM dNTPs (an equimolar mix of 25 mM dATP, dCTP, dGTP, and dTTP, Promega). .. The RNA templates were then degraded by adding 1 μl of 5 N NaOH and heating to 95° C for 3 min followed by a cooling to room temperature and neutralization with 1 μL of 5 N HCl.

Construct:

Article Title: The Staphylococcus aureus superantigen SElX is a bifunctional toxin that inhibits neutrophil functionStaphylococcal enterotoxin-like X (SElX) is a unique superantigen with functional features of two major families of staphylococcal virulence factors
Article Snippet: Site directed mutagenesis Site-directed mutagenesis was performed to exchange amino acids in the sequence of SElX with alanine by introducing mutations into the pET15b::selx2 construct using PCR with oligonucleotide primers listed in . .. Primers were used at a final concentration of 250 nM along with 2 mM dNTPs (Promega, Hampshire, UK).

Electrophoresis:

Article Title: Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications
Article Snippet: GsI-IIC RT (400 nM) was preincubated with the annealed R2/R2R-G heteroduplex (50 nM) and 50-nt acceptor template RNA (100 nM) in 10 μl of reaction medium containing 200 mM NaCl, 5 mM MgCl2 , 20 mM Tris-HCl pH 7.5, and 5 mM DTT for 30 min at room temperature, prior to initiating template-switching reverse transcription reactions by adding 0.4 μL of 25 mM dNTPs (an equimolar mix of 25 mM dATP, dCTP, dGTP, and dTTP, Promega). .. 10 μL of formamide loading dye (95% formamide, 0.025% xylene cyanol, 0.025% bromophenol blue, 6.25 mM EDTA) was added and products were denatured by heating to 99°C for 10 min and placed on ice prior to electrophoresis in a denaturing 8% TBE-urea polyacrylamide gel.

Incubation:

Article Title: Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications
Article Snippet: GsI-IIC RT (400 nM) was preincubated with the annealed R2/R2R-G heteroduplex (50 nM) and 50-nt acceptor template RNA (100 nM) in 10 μl of reaction medium containing 200 mM NaCl, 5 mM MgCl2 , 20 mM Tris-HCl pH 7.5, and 5 mM DTT for 30 min at room temperature, prior to initiating template-switching reverse transcription reactions by adding 0.4 μL of 25 mM dNTPs (an equimolar mix of 25 mM dATP, dCTP, dGTP, and dTTP, Promega). .. Reactions were incubated at 60° C for 15 min and stopped by adding 5 μL of the reaction mixture to 15 μL of 0.25 M EDTA.

Article Title: Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer
Article Snippet: .. The mixture was then used in a 20 μL reverse transcription reaction containing 200 μmol•L¯¹ each dNTPs and 20 U of reverse transcriptase (Promega), which was incubated at 37 °C for 1 h. The RNA-cDNA mixture (5 μL) was then used in 50 μL PCR reaction mixture containing all four dNTPs at 60 μmol•L¯¹, 5 U of Taq polymerase (Promega), and 50 pmol•L¯¹ of appropriate 5’ and 3’ primers[ , ]. .. VK1a and VK3a are 5’ primers for amplification of the κ chain with the Sac I site for cloning into the vector pComb3.

Article Title: The Staphylococcus aureus superantigen SElX is a bifunctional toxin that inhibits neutrophil functionStaphylococcal enterotoxin-like X (SElX) is a unique superantigen with functional features of two major families of staphylococcal virulence factors
Article Snippet: Primers were used at a final concentration of 250 nM along with 2 mM dNTPs (Promega, Hampshire, UK). .. Following the PCR reaction, Dpn I endonuclease (NEB, Herts, UK) was added to a final concentration of 0.8 U/μl, the reaction was then incubated for 1 h at 37°C followed by an enzyme deactivation step of 10 min at 65°C.

Article Title: Chronic hyperoxia alters the expression of neurotrophic factors in the carotid body of neonatal rats
Article Snippet: .. For BDNF, GDNF, and Ret, first strand cDNA was synthesized from 150 ng total RNA using 200U Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA), 1 μl primer mix (200 ng/μl oligo dT’s and 50 ng/μl random hexamers) (Promega, Madison, WI, USA), 1 μl 10 mM dNTPs (Promega), 4 μl 5× First strand buffer (Invitrogen), 2 μl 0.1 M dithiothreitol (DTT) (Invitrogen), and 1 μl RNAseOUT (Invitrogen); these reactions were incubated at 25°C for 5 min followed by 42°C for 50 min and 70°C for 15 min. For TrkB, Cbln1, and Fgf2, cDNA was synthesized from 200 ng total RNA using 1 μl ImProm-II Reverse Transcriptase (Promega), 1 μl primer mix (200 ng/μl oligo dT’s and 50 ng/μl random hexamers) (Promega), 1 μl 10 mM dNTPs (Promega), 4 μl ImProm-II 5× First strand buffer (Promega), 2 μl 25 mM MgCl2 (Promega), and 1 μl RNAseOUT (Invitrogen); these reactions were incubated at 25°C for 5 min followed by 42°C for 60 min and 70°C for 15 min. For Vgf, cDNA was synthesized from 300 ng total RNA using RT2 First Strand Kit (SABiosciences, Frederick, MD USA) according to kit instructions with the exception of replacing GE Buffer with water. .. The resulting single-stranded cDNA products were stored at −20°C until quantitative PCR analysis.

Mass Spectrometry:

Article Title: Pre-treatment or Post-treatment of Human Glioma Cells With BIX01294, the Inhibitor of Histone Methyltransferase G9a, Sensitizes Cells to Temozolomide
Article Snippet: Paragraph title: Bisulfite DNA Conversion and Methylation-Specific Polymerase Chain Reaction (MS-PCR) ... Each PCR mixture contained 1 μl of DNA, 500 nM of primers, 1x reaction buffer containing 1.5 mM MgCl2, 1 U HotStarTaq DNA Polymerase and 250 mM dNTPs (Promega, United States).

Modification:

Article Title: Pre-treatment or Post-treatment of Human Glioma Cells With BIX01294, the Inhibitor of Histone Methyltransferase G9a, Sensitizes Cells to Temozolomide
Article Snippet: The modified DNA was then amplified using primers specific for either methylated or unmethylated MGMT gene promoter sequences, as listed in Supplementary Table . .. Each PCR mixture contained 1 μl of DNA, 500 nM of primers, 1x reaction buffer containing 1.5 mM MgCl2, 1 U HotStarTaq DNA Polymerase and 250 mM dNTPs (Promega, United States).

Article Title: The First Identified Citrus tristeza virus Isolate of Turkey Contains a Mixture of Mild and Severe Strains
Article Snippet: Amplification of the CP and RdRp genes by RT-PCR dsRNA was isolated using a previously reported dsRNA isolation method ( ) with minor modification. .. First, 5 ml of dsRNA was denatured at 95ºC for 5 min and quickly chilled on ice. cDNA was synthesized from denatured dsRNA in 20 μl mixture containing 1X AMV first strand buffer (50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 ), 20 units RNAsin (Promega, USA), 0.5 mM dNTPs, 20 units AMV reverse transcriptase (Promega, USA) and 20 pmol random hexamers using the MJ Mini thermal cycler PTC1148 (Bio-Rad, USA) programmed at 30ºC for 10 min. followed by 42ºC for 60 min and 75ºC for 15 min. PCR was conducted in 50 μl reaction mixture containing 1X Pfu reaction buffer [20 mM Tris-HCl (pH 8.8 at 25°C), 10 mM (NH4 )2 SO4 , 100 mM KCl, 1% (v/v) Triton X-100, 1 mg/ml BSA and 20 mM MgSO4 ], 0.2 mM dNTPs, 2.5 unit Pfu DNA polymerase (Promega, USA) 5 μl of cDNA and 20 pmol of primer specific to CP (BC24 and 25) and RdRp (BC42 and BC43) genes.

Transformation Assay:

Article Title: The Staphylococcus aureus superantigen SElX is a bifunctional toxin that inhibits neutrophil functionStaphylococcal enterotoxin-like X (SElX) is a unique superantigen with functional features of two major families of staphylococcal virulence factors
Article Snippet: Primers were used at a final concentration of 250 nM along with 2 mM dNTPs (Promega, Hampshire, UK). .. Following digestion, 1 μl of the amplified vector product was transformed into E . coli Solopack cells from the Strataclone blunt cloning kit (Agilent technologies, UK) following the manufacturer’s instructions.

Flow Cytometry:

Article Title: Profiling proliferative cells and their progeny in damaged murine hearts
Article Snippet: .. DAPI-negative and MitoTracker-positive living cells were either sorted into TRIzol reagent (Thermo Scientific) for bulk mRNA sequencing or into 384-well plates containing 96 or 384 unique molecular identifier barcode primer sets, ERCC92 spike-ins (Agilent) and dNTPs (Promega) for single-cell mRNA-sequencing (SORT-seq) ( ) using a flow sorter (FACSAriaII, FACSFusion, or FACSJazz; all BD). ..

Reverse Transcription Polymerase Chain Reaction:

Article Title: CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes
Article Snippet: Paragraph title: Laser capture microdissection, RNA amplification, and reverse transcriptase-PCR (RT-PCR) ... A 50 μL PCR reaction was run with the following final concentrations: 450 ng of each primer (see for PCR primer sequences), 2 mM MgCl2 , 0.3 mM dNTPs, 2.5 U Taq polymerase (Promega GoFlex DNA polymerase) and 1 μL of 10 ng/μL DNA.

Article Title: The First Identified Citrus tristeza virus Isolate of Turkey Contains a Mixture of Mild and Severe Strains
Article Snippet: Paragraph title: Amplification of the CP and RdRp genes by RT-PCR ... First, 5 ml of dsRNA was denatured at 95ºC for 5 min and quickly chilled on ice. cDNA was synthesized from denatured dsRNA in 20 μl mixture containing 1X AMV first strand buffer (50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 ), 20 units RNAsin (Promega, USA), 0.5 mM dNTPs, 20 units AMV reverse transcriptase (Promega, USA) and 20 pmol random hexamers using the MJ Mini thermal cycler PTC1148 (Bio-Rad, USA) programmed at 30ºC for 10 min. followed by 42ºC for 60 min and 75ºC for 15 min. PCR was conducted in 50 μl reaction mixture containing 1X Pfu reaction buffer [20 mM Tris-HCl (pH 8.8 at 25°C), 10 mM (NH4 )2 SO4 , 100 mM KCl, 1% (v/v) Triton X-100, 1 mg/ml BSA and 20 mM MgSO4 ], 0.2 mM dNTPs, 2.5 unit Pfu DNA polymerase (Promega, USA) 5 μl of cDNA and 20 pmol of primer specific to CP (BC24 and 25) and RdRp (BC42 and BC43) genes.

Article Title: Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer
Article Snippet: Paragraph title: Amplifying Fd and κ chain genes of antibodies by RT-PCR ... The mixture was then used in a 20 μL reverse transcription reaction containing 200 μmol•L¯¹ each dNTPs and 20 U of reverse transcriptase (Promega), which was incubated at 37 °C for 1 h. The RNA-cDNA mixture (5 μL) was then used in 50 μL PCR reaction mixture containing all four dNTPs at 60 μmol•L¯¹, 5 U of Taq polymerase (Promega), and 50 pmol•L¯¹ of appropriate 5’ and 3’ primers[ , ].

Sequencing:

Article Title: Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications
Article Snippet: It consists of a 34-nt RNA oligonucleotide containing an Illumina R2 sequence (R2 RNA; ) with a 3′-blocking group (3SpC3; Integrated DNA Technologies) annealed to a 35-nt 5′ 32 P-labeled DNA primer ([γ-P32 ]-ATP, Perkin Elmer), which contains the reverse complement of the R2 sequence and leaves a single nucleotide 3′ G overhang (R2RG DNA; ). .. GsI-IIC RT (400 nM) was preincubated with the annealed R2/R2R-G heteroduplex (50 nM) and 50-nt acceptor template RNA (100 nM) in 10 μl of reaction medium containing 200 mM NaCl, 5 mM MgCl2 , 20 mM Tris-HCl pH 7.5, and 5 mM DTT for 30 min at room temperature, prior to initiating template-switching reverse transcription reactions by adding 0.4 μL of 25 mM dNTPs (an equimolar mix of 25 mM dATP, dCTP, dGTP, and dTTP, Promega).

Article Title: The Staphylococcus aureus superantigen SElX is a bifunctional toxin that inhibits neutrophil functionStaphylococcal enterotoxin-like X (SElX) is a unique superantigen with functional features of two major families of staphylococcal virulence factors
Article Snippet: Site directed mutagenesis Site-directed mutagenesis was performed to exchange amino acids in the sequence of SElX with alanine by introducing mutations into the pET15b::selx2 construct using PCR with oligonucleotide primers listed in . .. Primers were used at a final concentration of 250 nM along with 2 mM dNTPs (Promega, Hampshire, UK).

Article Title: Profiling proliferative cells and their progeny in damaged murine hearts
Article Snippet: .. DAPI-negative and MitoTracker-positive living cells were either sorted into TRIzol reagent (Thermo Scientific) for bulk mRNA sequencing or into 384-well plates containing 96 or 384 unique molecular identifier barcode primer sets, ERCC92 spike-ins (Agilent) and dNTPs (Promega) for single-cell mRNA-sequencing (SORT-seq) ( ) using a flow sorter (FACSAriaII, FACSFusion, or FACSJazz; all BD). ..

RNA Sequencing Assay:

Article Title: Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications
Article Snippet: The initial template-primer substrate used for template switching reactions was the same as that used for RNA-seq adapter addition in RNA-seq protocols ( ). .. GsI-IIC RT (400 nM) was preincubated with the annealed R2/R2R-G heteroduplex (50 nM) and 50-nt acceptor template RNA (100 nM) in 10 μl of reaction medium containing 200 mM NaCl, 5 mM MgCl2 , 20 mM Tris-HCl pH 7.5, and 5 mM DTT for 30 min at room temperature, prior to initiating template-switching reverse transcription reactions by adding 0.4 μL of 25 mM dNTPs (an equimolar mix of 25 mM dATP, dCTP, dGTP, and dTTP, Promega).

Methylation:

Article Title: Pre-treatment or Post-treatment of Human Glioma Cells With BIX01294, the Inhibitor of Histone Methyltransferase G9a, Sensitizes Cells to Temozolomide
Article Snippet: Paragraph title: Bisulfite DNA Conversion and Methylation-Specific Polymerase Chain Reaction (MS-PCR) ... Each PCR mixture contained 1 μl of DNA, 500 nM of primers, 1x reaction buffer containing 1.5 mM MgCl2, 1 U HotStarTaq DNA Polymerase and 250 mM dNTPs (Promega, United States).

Mutagenesis:

Article Title: The Staphylococcus aureus superantigen SElX is a bifunctional toxin that inhibits neutrophil functionStaphylococcal enterotoxin-like X (SElX) is a unique superantigen with functional features of two major families of staphylococcal virulence factors
Article Snippet: Paragraph title: Site directed mutagenesis ... Primers were used at a final concentration of 250 nM along with 2 mM dNTPs (Promega, Hampshire, UK).

Isolation:

Article Title: CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes
Article Snippet: Taste buds from the circumvallate papilla (CVP) and surrounding lingual epithelium (LE) were isolated using a Leica laser microdissection system LMD7000 (Leica). .. A 50 μL PCR reaction was run with the following final concentrations: 450 ng of each primer (see for PCR primer sequences), 2 mM MgCl2 , 0.3 mM dNTPs, 2.5 U Taq polymerase (Promega GoFlex DNA polymerase) and 1 μL of 10 ng/μL DNA.

Article Title: The First Identified Citrus tristeza virus Isolate of Turkey Contains a Mixture of Mild and Severe Strains
Article Snippet: Amplification of the CP and RdRp genes by RT-PCR dsRNA was isolated using a previously reported dsRNA isolation method ( ) with minor modification. .. First, 5 ml of dsRNA was denatured at 95ºC for 5 min and quickly chilled on ice. cDNA was synthesized from denatured dsRNA in 20 μl mixture containing 1X AMV first strand buffer (50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 ), 20 units RNAsin (Promega, USA), 0.5 mM dNTPs, 20 units AMV reverse transcriptase (Promega, USA) and 20 pmol random hexamers using the MJ Mini thermal cycler PTC1148 (Bio-Rad, USA) programmed at 30ºC for 10 min. followed by 42ºC for 60 min and 75ºC for 15 min. PCR was conducted in 50 μl reaction mixture containing 1X Pfu reaction buffer [20 mM Tris-HCl (pH 8.8 at 25°C), 10 mM (NH4 )2 SO4 , 100 mM KCl, 1% (v/v) Triton X-100, 1 mg/ml BSA and 20 mM MgSO4 ], 0.2 mM dNTPs, 2.5 unit Pfu DNA polymerase (Promega, USA) 5 μl of cDNA and 20 pmol of primer specific to CP (BC24 and 25) and RdRp (BC42 and BC43) genes.

Article Title: Chronic hyperoxia alters the expression of neurotrophic factors in the carotid body of neonatal rats
Article Snippet: Paragraph title: 2.2.2 RNA Isolation and cDNA Synthesis ... For BDNF, GDNF, and Ret, first strand cDNA was synthesized from 150 ng total RNA using 200U Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA), 1 μl primer mix (200 ng/μl oligo dT’s and 50 ng/μl random hexamers) (Promega, Madison, WI, USA), 1 μl 10 mM dNTPs (Promega), 4 μl 5× First strand buffer (Invitrogen), 2 μl 0.1 M dithiothreitol (DTT) (Invitrogen), and 1 μl RNAseOUT (Invitrogen); these reactions were incubated at 25°C for 5 min followed by 42°C for 50 min and 70°C for 15 min. For TrkB, Cbln1, and Fgf2, cDNA was synthesized from 200 ng total RNA using 1 μl ImProm-II Reverse Transcriptase (Promega), 1 μl primer mix (200 ng/μl oligo dT’s and 50 ng/μl random hexamers) (Promega), 1 μl 10 mM dNTPs (Promega), 4 μl ImProm-II 5× First strand buffer (Promega), 2 μl 25 mM MgCl2 (Promega), and 1 μl RNAseOUT (Invitrogen); these reactions were incubated at 25°C for 5 min followed by 42°C for 60 min and 70°C for 15 min. For Vgf, cDNA was synthesized from 300 ng total RNA using RT2 First Strand Kit (SABiosciences, Frederick, MD USA) according to kit instructions with the exception of replacing GE Buffer with water.

Size-exclusion Chromatography:

Article Title: CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes
Article Snippet: A 50 μL PCR reaction was run with the following final concentrations: 450 ng of each primer (see for PCR primer sequences), 2 mM MgCl2 , 0.3 mM dNTPs, 2.5 U Taq polymerase (Promega GoFlex DNA polymerase) and 1 μL of 10 ng/μL DNA. .. PCR cycling conditions used for Calhm1 were: 95°C for 3 min; 35 cycles of 95°C for 30 sec, 65°C for 30 sec, and 72°C for 45 sec; 72°C for 7 min; 4°C (hold).

Mouse Assay:

Article Title: CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes
Article Snippet: Taste buds and surrounding epithelium were pooled from a total of four mice of each genotype. .. A 50 μL PCR reaction was run with the following final concentrations: 450 ng of each primer (see for PCR primer sequences), 2 mM MgCl2 , 0.3 mM dNTPs, 2.5 U Taq polymerase (Promega GoFlex DNA polymerase) and 1 μL of 10 ng/μL DNA.

Polymerase Chain Reaction:

Article Title: CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes
Article Snippet: .. A 50 μL PCR reaction was run with the following final concentrations: 450 ng of each primer (see for PCR primer sequences), 2 mM MgCl2 , 0.3 mM dNTPs, 2.5 U Taq polymerase (Promega GoFlex DNA polymerase) and 1 μL of 10 ng/μL DNA. .. PCR cycling conditions used for Calhm1 were: 95°C for 3 min; 35 cycles of 95°C for 30 sec, 65°C for 30 sec, and 72°C for 45 sec; 72°C for 7 min; 4°C (hold).

Article Title: Pre-treatment or Post-treatment of Human Glioma Cells With BIX01294, the Inhibitor of Histone Methyltransferase G9a, Sensitizes Cells to Temozolomide
Article Snippet: .. Each PCR mixture contained 1 μl of DNA, 500 nM of primers, 1x reaction buffer containing 1.5 mM MgCl2, 1 U HotStarTaq DNA Polymerase and 250 mM dNTPs (Promega, United States). ..

Article Title: The First Identified Citrus tristeza virus Isolate of Turkey Contains a Mixture of Mild and Severe Strains
Article Snippet: .. First, 5 ml of dsRNA was denatured at 95ºC for 5 min and quickly chilled on ice. cDNA was synthesized from denatured dsRNA in 20 μl mixture containing 1X AMV first strand buffer (50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 ), 20 units RNAsin (Promega, USA), 0.5 mM dNTPs, 20 units AMV reverse transcriptase (Promega, USA) and 20 pmol random hexamers using the MJ Mini thermal cycler PTC1148 (Bio-Rad, USA) programmed at 30ºC for 10 min. followed by 42ºC for 60 min and 75ºC for 15 min. PCR was conducted in 50 μl reaction mixture containing 1X Pfu reaction buffer [20 mM Tris-HCl (pH 8.8 at 25°C), 10 mM (NH4 )2 SO4 , 100 mM KCl, 1% (v/v) Triton X-100, 1 mg/ml BSA and 20 mM MgSO4 ], 0.2 mM dNTPs, 2.5 unit Pfu DNA polymerase (Promega, USA) 5 μl of cDNA and 20 pmol of primer specific to CP (BC24 and 25) and RdRp (BC42 and BC43) genes. .. PCR was performed in the MJ Mini thermal cycler PTC1148 (Bio-Rad, USA) programmed for initial denaturation at 94°C for 3 min and then, 40 cycles of denaturation at 94°C for 30 s, primer annealing at 55°C for 30 s, and primer extension at 72°C for 1 min for the CP gene and 2 min for the RdRp gene followed by final extension at 72°C for 5 min. PCR products were separated in 1% agarose gel with 100 bp DNA size marker, stained with ethidium bromide and visualized and analyzed by Doc-It system (UVP, England).

Article Title: Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer
Article Snippet: .. The mixture was then used in a 20 μL reverse transcription reaction containing 200 μmol•L¯¹ each dNTPs and 20 U of reverse transcriptase (Promega), which was incubated at 37 °C for 1 h. The RNA-cDNA mixture (5 μL) was then used in 50 μL PCR reaction mixture containing all four dNTPs at 60 μmol•L¯¹, 5 U of Taq polymerase (Promega), and 50 pmol•L¯¹ of appropriate 5’ and 3’ primers[ , ]. .. VK1a and VK3a are 5’ primers for amplification of the κ chain with the Sac I site for cloning into the vector pComb3.

Article Title: The Staphylococcus aureus superantigen SElX is a bifunctional toxin that inhibits neutrophil functionStaphylococcal enterotoxin-like X (SElX) is a unique superantigen with functional features of two major families of staphylococcal virulence factors
Article Snippet: Site directed mutagenesis Site-directed mutagenesis was performed to exchange amino acids in the sequence of SElX with alanine by introducing mutations into the pET15b::selx2 construct using PCR with oligonucleotide primers listed in . .. Primers were used at a final concentration of 250 nM along with 2 mM dNTPs (Promega, Hampshire, UK).

Article Title: Knockdown of FBXO39 inhibits proliferation and promotes apoptosis of human osteosarcoma U-2OS cells
Article Snippet: Paragraph title: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis ... Reverse transcription was performed using M-MLV Reverse Transcriptase, RNase Inhibitor and dNTPs (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol.

Quantitative RT-PCR:

Article Title: Knockdown of FBXO39 inhibits proliferation and promotes apoptosis of human osteosarcoma U-2OS cells
Article Snippet: Paragraph title: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis ... Reverse transcription was performed using M-MLV Reverse Transcriptase, RNase Inhibitor and dNTPs (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol.

Staining:

Article Title: The First Identified Citrus tristeza virus Isolate of Turkey Contains a Mixture of Mild and Severe Strains
Article Snippet: First, 5 ml of dsRNA was denatured at 95ºC for 5 min and quickly chilled on ice. cDNA was synthesized from denatured dsRNA in 20 μl mixture containing 1X AMV first strand buffer (50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 ), 20 units RNAsin (Promega, USA), 0.5 mM dNTPs, 20 units AMV reverse transcriptase (Promega, USA) and 20 pmol random hexamers using the MJ Mini thermal cycler PTC1148 (Bio-Rad, USA) programmed at 30ºC for 10 min. followed by 42ºC for 60 min and 75ºC for 15 min. PCR was conducted in 50 μl reaction mixture containing 1X Pfu reaction buffer [20 mM Tris-HCl (pH 8.8 at 25°C), 10 mM (NH4 )2 SO4 , 100 mM KCl, 1% (v/v) Triton X-100, 1 mg/ml BSA and 20 mM MgSO4 ], 0.2 mM dNTPs, 2.5 unit Pfu DNA polymerase (Promega, USA) 5 μl of cDNA and 20 pmol of primer specific to CP (BC24 and 25) and RdRp (BC42 and BC43) genes. .. PCR was performed in the MJ Mini thermal cycler PTC1148 (Bio-Rad, USA) programmed for initial denaturation at 94°C for 3 min and then, 40 cycles of denaturation at 94°C for 30 s, primer annealing at 55°C for 30 s, and primer extension at 72°C for 1 min for the CP gene and 2 min for the RdRp gene followed by final extension at 72°C for 5 min. PCR products were separated in 1% agarose gel with 100 bp DNA size marker, stained with ethidium bromide and visualized and analyzed by Doc-It system (UVP, England).

Purification:

Article Title: CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes
Article Snippet: Total RNA from taste bud and LE samples was purified using a RNAqueous-Micro Kit (Ambion). .. A 50 μL PCR reaction was run with the following final concentrations: 450 ng of each primer (see for PCR primer sequences), 2 mM MgCl2 , 0.3 mM dNTPs, 2.5 U Taq polymerase (Promega GoFlex DNA polymerase) and 1 μL of 10 ng/μL DNA.

Article Title: Profiling proliferative cells and their progeny in damaged murine hearts
Article Snippet: Paragraph title: Flow Cytometric Purification. ... DAPI-negative and MitoTracker-positive living cells were either sorted into TRIzol reagent (Thermo Scientific) for bulk mRNA sequencing or into 384-well plates containing 96 or 384 unique molecular identifier barcode primer sets, ERCC92 spike-ins (Agilent) and dNTPs (Promega) for single-cell mRNA-sequencing (SORT-seq) ( ) using a flow sorter (FACSAriaII, FACSFusion, or FACSJazz; all BD).

Chromatin Immunoprecipitation:

Article Title: Chronic hyperoxia alters the expression of neurotrophic factors in the carotid body of neonatal rats
Article Snippet: RNA quantity and quality were assessed using an RNA 6000 Nano Chip in a Bioanalyzer (Agilent, Santa Clara, CA, USA). .. For BDNF, GDNF, and Ret, first strand cDNA was synthesized from 150 ng total RNA using 200U Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA), 1 μl primer mix (200 ng/μl oligo dT’s and 50 ng/μl random hexamers) (Promega, Madison, WI, USA), 1 μl 10 mM dNTPs (Promega), 4 μl 5× First strand buffer (Invitrogen), 2 μl 0.1 M dithiothreitol (DTT) (Invitrogen), and 1 μl RNAseOUT (Invitrogen); these reactions were incubated at 25°C for 5 min followed by 42°C for 50 min and 70°C for 15 min. For TrkB, Cbln1, and Fgf2, cDNA was synthesized from 200 ng total RNA using 1 μl ImProm-II Reverse Transcriptase (Promega), 1 μl primer mix (200 ng/μl oligo dT’s and 50 ng/μl random hexamers) (Promega), 1 μl 10 mM dNTPs (Promega), 4 μl ImProm-II 5× First strand buffer (Promega), 2 μl 25 mM MgCl2 (Promega), and 1 μl RNAseOUT (Invitrogen); these reactions were incubated at 25°C for 5 min followed by 42°C for 60 min and 70°C for 15 min. For Vgf, cDNA was synthesized from 300 ng total RNA using RT2 First Strand Kit (SABiosciences, Frederick, MD USA) according to kit instructions with the exception of replacing GE Buffer with water.

SDS Page:

Article Title: Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications
Article Snippet: Fractions containing GsI-IIC RT were identified by SDS-PAGE, pooled, and dialyzed into 20 mM Tris-HCl pH 7.5, 500 mM KCl, 50% glycerol. .. GsI-IIC RT (400 nM) was preincubated with the annealed R2/R2R-G heteroduplex (50 nM) and 50-nt acceptor template RNA (100 nM) in 10 μl of reaction medium containing 200 mM NaCl, 5 mM MgCl2 , 20 mM Tris-HCl pH 7.5, and 5 mM DTT for 30 min at room temperature, prior to initiating template-switching reverse transcription reactions by adding 0.4 μL of 25 mM dNTPs (an equimolar mix of 25 mM dATP, dCTP, dGTP, and dTTP, Promega).

Plasmid Preparation:

Article Title: Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer
Article Snippet: The mixture was then used in a 20 μL reverse transcription reaction containing 200 μmol•L¯¹ each dNTPs and 20 U of reverse transcriptase (Promega), which was incubated at 37 °C for 1 h. The RNA-cDNA mixture (5 μL) was then used in 50 μL PCR reaction mixture containing all four dNTPs at 60 μmol•L¯¹, 5 U of Taq polymerase (Promega), and 50 pmol•L¯¹ of appropriate 5’ and 3’ primers[ , ]. .. VK1a and VK3a are 5’ primers for amplification of the κ chain with the Sac I site for cloning into the vector pComb3.

Article Title: The Staphylococcus aureus superantigen SElX is a bifunctional toxin that inhibits neutrophil functionStaphylococcal enterotoxin-like X (SElX) is a unique superantigen with functional features of two major families of staphylococcal virulence factors
Article Snippet: Primers were used at a final concentration of 250 nM along with 2 mM dNTPs (Promega, Hampshire, UK). .. Following digestion, 1 μl of the amplified vector product was transformed into E . coli Solopack cells from the Strataclone blunt cloning kit (Agilent technologies, UK) following the manufacturer’s instructions.

Software:

Article Title: Homeostatic regulation of T cell trafficking by a B cell derived peptide is impaired in autoimmune and chronic inflammatory disease
Article Snippet: To convert mRNA to cDNA, random primers (Promega, Maddison, USA) were annealed to 1 μg of mRNA for 5 minutes at 70°C, after which the following mastermix was added to give a final volume of 30 μl: 10 U Superscript II Reverse Transcriptase (RT), 10 U RNAout RNase inhibitor, 1X Superscript Buffer (all from Invitrogen) and 10 mM dNTPs (Promega). .. Samples were amplified in duplicates using the 7500HT Real-Time PCR machine (Applied Biosystems) and analyzed using the software package SDS 2.2 (Applied Biosystems).

Real-time Polymerase Chain Reaction:

Article Title: Knockdown of FBXO39 inhibits proliferation and promotes apoptosis of human osteosarcoma U-2OS cells
Article Snippet: Reverse transcription was performed using M-MLV Reverse Transcriptase, RNase Inhibitor and dNTPs (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol. .. RT-qPCR was performed using SYBR Master mix (Takara Biotechnology Co., Ltd., Dalian) and a Roche Light Cycler 480 Real-time PCR system (Roche Diagnostics, Basel, Switzerland).

Article Title: Homeostatic regulation of T cell trafficking by a B cell derived peptide is impaired in autoimmune and chronic inflammatory disease
Article Snippet: Paragraph title: Real-time PCR ... To convert mRNA to cDNA, random primers (Promega, Maddison, USA) were annealed to 1 μg of mRNA for 5 minutes at 70°C, after which the following mastermix was added to give a final volume of 30 μl: 10 U Superscript II Reverse Transcriptase (RT), 10 U RNAout RNase inhibitor, 1X Superscript Buffer (all from Invitrogen) and 10 mM dNTPs (Promega).

Article Title: Chronic hyperoxia alters the expression of neurotrophic factors in the carotid body of neonatal rats
Article Snippet: For BDNF, GDNF, and Ret, first strand cDNA was synthesized from 150 ng total RNA using 200U Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA), 1 μl primer mix (200 ng/μl oligo dT’s and 50 ng/μl random hexamers) (Promega, Madison, WI, USA), 1 μl 10 mM dNTPs (Promega), 4 μl 5× First strand buffer (Invitrogen), 2 μl 0.1 M dithiothreitol (DTT) (Invitrogen), and 1 μl RNAseOUT (Invitrogen); these reactions were incubated at 25°C for 5 min followed by 42°C for 50 min and 70°C for 15 min. For TrkB, Cbln1, and Fgf2, cDNA was synthesized from 200 ng total RNA using 1 μl ImProm-II Reverse Transcriptase (Promega), 1 μl primer mix (200 ng/μl oligo dT’s and 50 ng/μl random hexamers) (Promega), 1 μl 10 mM dNTPs (Promega), 4 μl ImProm-II 5× First strand buffer (Promega), 2 μl 25 mM MgCl2 (Promega), and 1 μl RNAseOUT (Invitrogen); these reactions were incubated at 25°C for 5 min followed by 42°C for 60 min and 70°C for 15 min. For Vgf, cDNA was synthesized from 300 ng total RNA using RT2 First Strand Kit (SABiosciences, Frederick, MD USA) according to kit instructions with the exception of replacing GE Buffer with water. .. The resulting single-stranded cDNA products were stored at −20°C until quantitative PCR analysis.

Agarose Gel Electrophoresis:

Article Title: Pre-treatment or Post-treatment of Human Glioma Cells With BIX01294, the Inhibitor of Histone Methyltransferase G9a, Sensitizes Cells to Temozolomide
Article Snippet: Each PCR mixture contained 1 μl of DNA, 500 nM of primers, 1x reaction buffer containing 1.5 mM MgCl2, 1 U HotStarTaq DNA Polymerase and 250 mM dNTPs (Promega, United States). .. The PCR products were visualized using 1.5% agarose gel or Agilent Bioanalyzer yielding a band of 81 bp for a methylated and 93 bp for an unmethylated product.

Article Title: The First Identified Citrus tristeza virus Isolate of Turkey Contains a Mixture of Mild and Severe Strains
Article Snippet: First, 5 ml of dsRNA was denatured at 95ºC for 5 min and quickly chilled on ice. cDNA was synthesized from denatured dsRNA in 20 μl mixture containing 1X AMV first strand buffer (50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 ), 20 units RNAsin (Promega, USA), 0.5 mM dNTPs, 20 units AMV reverse transcriptase (Promega, USA) and 20 pmol random hexamers using the MJ Mini thermal cycler PTC1148 (Bio-Rad, USA) programmed at 30ºC for 10 min. followed by 42ºC for 60 min and 75ºC for 15 min. PCR was conducted in 50 μl reaction mixture containing 1X Pfu reaction buffer [20 mM Tris-HCl (pH 8.8 at 25°C), 10 mM (NH4 )2 SO4 , 100 mM KCl, 1% (v/v) Triton X-100, 1 mg/ml BSA and 20 mM MgSO4 ], 0.2 mM dNTPs, 2.5 unit Pfu DNA polymerase (Promega, USA) 5 μl of cDNA and 20 pmol of primer specific to CP (BC24 and 25) and RdRp (BC42 and BC43) genes. .. PCR was performed in the MJ Mini thermal cycler PTC1148 (Bio-Rad, USA) programmed for initial denaturation at 94°C for 3 min and then, 40 cycles of denaturation at 94°C for 30 s, primer annealing at 55°C for 30 s, and primer extension at 72°C for 1 min for the CP gene and 2 min for the RdRp gene followed by final extension at 72°C for 5 min. PCR products were separated in 1% agarose gel with 100 bp DNA size marker, stained with ethidium bromide and visualized and analyzed by Doc-It system (UVP, England).

Laser Capture Microdissection:

Article Title: CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes
Article Snippet: Paragraph title: Laser capture microdissection, RNA amplification, and reverse transcriptase-PCR (RT-PCR) ... A 50 μL PCR reaction was run with the following final concentrations: 450 ng of each primer (see for PCR primer sequences), 2 mM MgCl2 , 0.3 mM dNTPs, 2.5 U Taq polymerase (Promega GoFlex DNA polymerase) and 1 μL of 10 ng/μL DNA.

Concentration Assay:

Article Title: The Staphylococcus aureus superantigen SElX is a bifunctional toxin that inhibits neutrophil functionStaphylococcal enterotoxin-like X (SElX) is a unique superantigen with functional features of two major families of staphylococcal virulence factors
Article Snippet: .. Primers were used at a final concentration of 250 nM along with 2 mM dNTPs (Promega, Hampshire, UK). .. PCR cycle conditions were as follows; 1 cycle at 95°C for 2 min, 30 cycles of 95°C for 20 s, 50°C for 20 s and 72°C for 90 s, followed by a final extension of 3 min at 72°C.

Article Title: Homeostatic regulation of T cell trafficking by a B cell derived peptide is impaired in autoimmune and chronic inflammatory disease
Article Snippet: Briefly, PBMC were first lysed, then added to a column, after three washes, mRNA was eluted from the column with water. mRNA concentration was measured using Nanodrop spectrofluorimeter (LabTech) and mRNA was stored at −80°C. .. To convert mRNA to cDNA, random primers (Promega, Maddison, USA) were annealed to 1 μg of mRNA for 5 minutes at 70°C, after which the following mastermix was added to give a final volume of 30 μl: 10 U Superscript II Reverse Transcriptase (RT), 10 U RNAout RNase inhibitor, 1X Superscript Buffer (all from Invitrogen) and 10 mM dNTPs (Promega).

Marker:

Article Title: The First Identified Citrus tristeza virus Isolate of Turkey Contains a Mixture of Mild and Severe Strains
Article Snippet: First, 5 ml of dsRNA was denatured at 95ºC for 5 min and quickly chilled on ice. cDNA was synthesized from denatured dsRNA in 20 μl mixture containing 1X AMV first strand buffer (50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 ), 20 units RNAsin (Promega, USA), 0.5 mM dNTPs, 20 units AMV reverse transcriptase (Promega, USA) and 20 pmol random hexamers using the MJ Mini thermal cycler PTC1148 (Bio-Rad, USA) programmed at 30ºC for 10 min. followed by 42ºC for 60 min and 75ºC for 15 min. PCR was conducted in 50 μl reaction mixture containing 1X Pfu reaction buffer [20 mM Tris-HCl (pH 8.8 at 25°C), 10 mM (NH4 )2 SO4 , 100 mM KCl, 1% (v/v) Triton X-100, 1 mg/ml BSA and 20 mM MgSO4 ], 0.2 mM dNTPs, 2.5 unit Pfu DNA polymerase (Promega, USA) 5 μl of cDNA and 20 pmol of primer specific to CP (BC24 and 25) and RdRp (BC42 and BC43) genes. .. PCR was performed in the MJ Mini thermal cycler PTC1148 (Bio-Rad, USA) programmed for initial denaturation at 94°C for 3 min and then, 40 cycles of denaturation at 94°C for 30 s, primer annealing at 55°C for 30 s, and primer extension at 72°C for 1 min for the CP gene and 2 min for the RdRp gene followed by final extension at 72°C for 5 min. PCR products were separated in 1% agarose gel with 100 bp DNA size marker, stained with ethidium bromide and visualized and analyzed by Doc-It system (UVP, England).

Lysis:

Article Title: Chronic hyperoxia alters the expression of neurotrophic factors in the carotid body of neonatal rats
Article Snippet: In each of the three experiments, pooled carotid bodies were homogenized for 20 s in RLT lysis buffer (Qiagen, Valencia, CA, USA), and RNA was extracted from the homogenates using an RNeasy Micro RNA Isolation Kit (Qiagen). .. For BDNF, GDNF, and Ret, first strand cDNA was synthesized from 150 ng total RNA using 200U Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA), 1 μl primer mix (200 ng/μl oligo dT’s and 50 ng/μl random hexamers) (Promega, Madison, WI, USA), 1 μl 10 mM dNTPs (Promega), 4 μl 5× First strand buffer (Invitrogen), 2 μl 0.1 M dithiothreitol (DTT) (Invitrogen), and 1 μl RNAseOUT (Invitrogen); these reactions were incubated at 25°C for 5 min followed by 42°C for 50 min and 70°C for 15 min. For TrkB, Cbln1, and Fgf2, cDNA was synthesized from 200 ng total RNA using 1 μl ImProm-II Reverse Transcriptase (Promega), 1 μl primer mix (200 ng/μl oligo dT’s and 50 ng/μl random hexamers) (Promega), 1 μl 10 mM dNTPs (Promega), 4 μl ImProm-II 5× First strand buffer (Promega), 2 μl 25 mM MgCl2 (Promega), and 1 μl RNAseOUT (Invitrogen); these reactions were incubated at 25°C for 5 min followed by 42°C for 60 min and 70°C for 15 min. For Vgf, cDNA was synthesized from 300 ng total RNA using RT2 First Strand Kit (SABiosciences, Frederick, MD USA) according to kit instructions with the exception of replacing GE Buffer with water.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    Promega dntp master mix
    Dntp Master Mix, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dntp master mix/product/Promega
    Average 90 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    dntp master mix - by Bioz Stars, 2020-01
    90/100 stars
      Buy from Supplier

    90
    Promega deoxynucleotide triphosphates dntps
    Deoxynucleotide Triphosphates Dntps, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1807 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/deoxynucleotide triphosphates dntps/product/Promega
    Average 90 stars, based on 1807 article reviews
    Price from $9.99 to $1999.99
    deoxynucleotide triphosphates dntps - by Bioz Stars, 2020-01
    90/100 stars
      Buy from Supplier

    Image Search Results