Structured Review

Promega dntps
In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. <t>bRT</t> is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and <t>dNTPs,</t> including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.
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Images

1) Product Images from "Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex"

Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky620

In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.
Figure Legend Snippet: In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.

Techniques Used: In Vitro, Binding Assay, Sequencing, Incubation, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Activity Assay, Produced

Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.
Figure Legend Snippet: Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.

Techniques Used: Activity Assay, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Incubation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.
Figure Legend Snippet: Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

Techniques Used: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Amplification, Produced, Sequencing, Activity Assay, Polyacrylamide Gel Electrophoresis

2) Product Images from "Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA"

Article Title: Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA

Journal: ACS Chemical Biology

doi: 10.1021/cb500270x

Controlling the extent of pausing by AMV-RT at a 7-EAA site. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide. (B) Primer extension results for reactions containing 10 μM dNTPs at 42 °C for 45 min. Lanes are labeled as follows for N: −, labeled primer only, no extension; A, adenosine; 7, 7-EAA; B, biotin triazole; and B + S, biotin triazole + monomeric streptavidin. (C) Primer extension results for reactions containing 1 μM dNTPs at 37 °C for 5 min; lanes are labeled the same as in panel B, and the arrow indicates a pause site. (D) Quantification of inhibition of primer extension under the conditions used in panel C; the average for at least three independent primer extension reactions ± standard deviation is plotted.
Figure Legend Snippet: Controlling the extent of pausing by AMV-RT at a 7-EAA site. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide. (B) Primer extension results for reactions containing 10 μM dNTPs at 42 °C for 45 min. Lanes are labeled as follows for N: −, labeled primer only, no extension; A, adenosine; 7, 7-EAA; B, biotin triazole; and B + S, biotin triazole + monomeric streptavidin. (C) Primer extension results for reactions containing 1 μM dNTPs at 37 °C for 5 min; lanes are labeled the same as in panel B, and the arrow indicates a pause site. (D) Quantification of inhibition of primer extension under the conditions used in panel C; the average for at least three independent primer extension reactions ± standard deviation is plotted.

Techniques Used: Primer Extension Assay, Labeling, Inhibition, Standard Deviation

3) 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

4) Product Images from "Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA"

Article Title: Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA

Journal: ACS Chemical Biology

doi: 10.1021/cb500270x

Controlling the extent of pausing by AMV-RT at a 7-EAA site. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide. (B) Primer extension results for reactions containing 10 μM dNTPs at 42 °C for 45 min. Lanes are labeled as follows for N: −, labeled primer only, no extension; A, adenosine; 7, 7-EAA; B, biotin triazole; and B + S, biotin triazole + monomeric streptavidin. (C) Primer extension results for reactions containing 1 μM dNTPs at 37 °C for 5 min; lanes are labeled the same as in panel B, and the arrow indicates a pause site. (D) Quantification of inhibition of primer extension under the conditions used in panel C; the average for at least three independent primer extension reactions ± standard deviation is plotted.
Figure Legend Snippet: Controlling the extent of pausing by AMV-RT at a 7-EAA site. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide. (B) Primer extension results for reactions containing 10 μM dNTPs at 42 °C for 45 min. Lanes are labeled as follows for N: −, labeled primer only, no extension; A, adenosine; 7, 7-EAA; B, biotin triazole; and B + S, biotin triazole + monomeric streptavidin. (C) Primer extension results for reactions containing 1 μM dNTPs at 37 °C for 5 min; lanes are labeled the same as in panel B, and the arrow indicates a pause site. (D) Quantification of inhibition of primer extension under the conditions used in panel C; the average for at least three independent primer extension reactions ± standard deviation is plotted.

Techniques Used: Primer Extension Assay, Labeling, Inhibition, Standard Deviation

Related Articles

Clone Assay:

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Article Snippet: Blackcurrant cDNA inserts were amplified by PCR using plasmid DNA template and M13 forward and reverse primers that span the multiple cloning site of the vector. .. Each reaction was performed in 100 μl containing 50 ng of plasmid, M13 primers (0.5 μM each), 0.2 mM dNTPs, 2 mM dNTPs, 2 mM MgCl2 and 0.10 U/μl Taq DNA Polymerase (Promega) in 1 × PCR buffer.

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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.

Centrifugation:

Article Title: Biofilm Formation by ica-Negative Ocular Isolates of Staphylococcus haemolyticus
Article Snippet: The next day, the ethanol precipitated DNA was collected by centrifugation for 20 min at 4°C and 18,000 × g , washed with ice-cold 70% ethanol, air dried, and dissolved in 30 μl of nuclease-free water. .. PCR mixture contained: 4 μl of 5× PCR buffer, 1 μl of 25 mM MgCl2 , 2 μl each of dNTPs (Promega, Madison, WI, United States), 2 μl each of forward and reverse primer (5 pmol/μl) (GCC Biotech, New Delhi), 0.25 μl of 5 U/μl Taq polymerase (Promega, Madison, WI, United States), 1 μl of diluted eDNA, and 7.75 μl of nuclease-free water.

Amplification:

Article Title: Candidate genes associated with bud dormancy release in blackcurrant (Ribes nigrum L.)
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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).

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).

Synthesized:

Article Title: Chronic hyperoxia alters the expression of neurotrophic factors in the carotid body of neonatal rats
Article Snippet: Total RNA yield averaged 0.6 μg per sample (30 ng per carotid body). .. 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.

Article Title: Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted anti-viral functions of APOBEC3G
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Construct:

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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).

Real-time Polymerase Chain Reaction:

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: The effect of centromere protein U silencing by lentiviral mediated RNA interference on the proliferation and apoptosis of breast cancer
Article Snippet: Then, a RT reaction system, including 5×RT buffer (4.0 µl), RNA-free H2 O (2.6 µl), 10 mM dNTPs (2.0 µl; Promega, USA), RNasin (0.4 µl) and M-MLV-RTase (1.0 µl), was run for 1 h at 42°C, then transferred to a 70°C water bath for 10 min to inactivate the RTase. .. PCR was performed using 1.0 µl cDNA with SYBRGreen PCR Master mix kit (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocol.

Microarray:

Article Title: Candidate genes associated with bud dormancy release in blackcurrant (Ribes nigrum L.)
Article Snippet: Paragraph title: Microarray fabrication ... Each reaction was performed in 100 μl containing 50 ng of plasmid, M13 primers (0.5 μM each), 0.2 mM dNTPs, 2 mM dNTPs, 2 mM MgCl2 and 0.10 U/μl Taq DNA Polymerase (Promega) in 1 × PCR buffer.

Incubation:

Article Title: Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA
Article Snippet: RNA template (40 nM) was incubated with ∼160 nM 32 P-labeled GluR B pre-mRNA primer and 1× Promega AMV buffer for 15 min at 62 °C. .. Samples were then mixed with AMV-RT and dNTP mix so that the final concentrations were as follows: 20 nM RNA, ∼80 nM 32 P-labeled GluR B pre-mRNA 18 nt primer, 10 μM dNTPs, 1× Promega AMV-RT buffer, and 5 units of AMV-RT for the standard extension conditions protocol and 20 nM RNA, ∼80 nM 32 P-labeled GluR B pre-mRNA 18 nt primer, 1 μM dNTPs, 1× Promega AMV-RT buffer, and 5 units of AMV-RT for the low [dNTP] conditions protocol.

Article Title: Chronic hyperoxia alters the expression of neurotrophic factors in the carotid body of neonatal rats
Article Snippet: Total RNA yield averaged 0.6 μg per sample (30 ng per carotid body). .. 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.

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: Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer
Article Snippet: Total RNA (20-50 μg) was added to 60 pmol primer of Oligo (dt) and heated at 65 °C for 10 min. .. 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: Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted anti-viral functions of APOBEC3G
Article Snippet: Reactions of 10 μl were prepared in this buffer with 10 nM template, 200 μM dNTPs and 1 U/μl RNAase Inhibitor (Promega). .. Reactions were preincubated for 10 min at 37 °C with indicated amounts of purified A3G (dialyzed against 50 mM Tris HCL pH 8.9, 150 mM NaCl, 10% glycerol, 0.1% NP40, 1 mM DTT) or dialysis buffer alone.

Formalin-fixed Paraffin-Embedded:

Article Title: Analysis of Zinc-Exporters Expression in Prostate Cancer
Article Snippet: The corresponding benign and tumor areas on unstained slides were scratched out with sterile surgical blades and processed for RNA isolation using RNeasy formalin fixed paraffin-embedded (FFPE) kit (Qiagen) per manufacturer’s protocol. .. First strand cDNA was transcribed with random primers, dNTPs and M-MLV reverse transcriptase (Promega).

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). .. 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).

Modification:

Article Title: Candidate genes associated with bud dormancy release in blackcurrant (Ribes nigrum L.)
Article Snippet: Each reaction was performed in 100 μl containing 50 ng of plasmid, M13 primers (0.5 μM each), 0.2 mM dNTPs, 2 mM dNTPs, 2 mM MgCl2 and 0.10 U/μl Taq DNA Polymerase (Promega) in 1 × PCR buffer. .. Each reaction was performed in 100 μl containing 50 ng of plasmid, M13 primers (0.5 μM each), 0.2 mM dNTPs, 2 mM dNTPs, 2 mM MgCl2 and 0.10 U/μl Taq DNA Polymerase (Promega) in 1 × PCR buffer.

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: Analysis of Zinc-Exporters Expression in Prostate Cancer
Article Snippet: This kit is designed for efficiently extracting RNA from tissue sections by reversing formaldehyde modification of RNA. .. First strand cDNA was transcribed with random primers, dNTPs and M-MLV reverse transcriptase (Promega).

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). .. Primers were used at a final concentration of 250 nM along with 2 mM dNTPs (Promega, Hampshire, UK).

Flow Cytometry:

Article Title: Profiling proliferative cells and their progeny in damaged murine hearts
Article Snippet: 4′,6-Diamidino-2-phenylindole (DAPI) was added immediately before flow sorting. .. 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). .. Samples were lysed and RNA from each bulk sort sample or single cell was barcoded and processed using the CEL-Seq2 technique ( , , ).

Sequencing:

Article Title: Profiling proliferative cells and their progeny in damaged murine hearts
Article Snippet: 4′,6-Diamidino-2-phenylindole (DAPI) was added immediately before flow sorting. .. 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). .. Samples were lysed and RNA from each bulk sort sample or single cell was barcoded and processed using the CEL-Seq2 technique ( , , ).

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: Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted anti-viral functions of APOBEC3G
Article Snippet: Reactions of 10 μl were prepared in this buffer with 10 nM template, 200 μM dNTPs and 1 U/μl RNAase Inhibitor (Promega). .. Unless otherwise noted, the reactions were initiated by addition of 10 nM HIV-1 RT (Worthington), and incubated for the indicated amount of time at 37°C.

Imaging:

Article Title: Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA
Article Snippet: Samples were then mixed with AMV-RT and dNTP mix so that the final concentrations were as follows: 20 nM RNA, ∼80 nM 32 P-labeled GluR B pre-mRNA 18 nt primer, 10 μM dNTPs, 1× Promega AMV-RT buffer, and 5 units of AMV-RT for the standard extension conditions protocol and 20 nM RNA, ∼80 nM 32 P-labeled GluR B pre-mRNA 18 nt primer, 1 μM dNTPs, 1× Promega AMV-RT buffer, and 5 units of AMV-RT for the low [dNTP] conditions protocol. .. Samples were then mixed with AMV-RT and dNTP mix so that the final concentrations were as follows: 20 nM RNA, ∼80 nM 32 P-labeled GluR B pre-mRNA 18 nt primer, 10 μM dNTPs, 1× Promega AMV-RT buffer, and 5 units of AMV-RT for the standard extension conditions protocol and 20 nM RNA, ∼80 nM 32 P-labeled GluR B pre-mRNA 18 nt primer, 1 μM dNTPs, 1× Promega AMV-RT buffer, and 5 units of AMV-RT for the low [dNTP] conditions protocol.

Article Title: Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA
Article Snippet: For each reaction, dNTPs, ddNTPs, 5× Promega AMV-RT buffer, and AMV-RT were added so that the concentrations in the reaction were as follows: 20 nM RNA, ∼80 nM 32 P-labeled 21 nt GluR B pre-mRNA primer, 10 mM of the 3 dNTPs, 10 mM of ddNTP for the fourth base, 1× Promega AMV-RT buffer, and 5 units of AMV-RT. .. For each reaction, dNTPs, ddNTPs, 5× Promega AMV-RT buffer, and AMV-RT were added so that the concentrations in the reaction were as follows: 20 nM RNA, ∼80 nM 32 P-labeled 21 nt GluR B pre-mRNA primer, 10 mM of the 3 dNTPs, 10 mM of ddNTP for the fourth base, 1× Promega AMV-RT buffer, and 5 units of AMV-RT.

Polymerase Chain Reaction:

Article Title: Candidate genes associated with bud dormancy release in blackcurrant (Ribes nigrum L.)
Article Snippet: Blackcurrant cDNA inserts were amplified by PCR using plasmid DNA template and M13 forward and reverse primers that span the multiple cloning site of the vector. .. Each reaction was performed in 100 μl containing 50 ng of plasmid, M13 primers (0.5 μM each), 0.2 mM dNTPs, 2 mM dNTPs, 2 mM MgCl2 and 0.10 U/μl Taq DNA Polymerase (Promega) in 1 × PCR buffer. .. PCR conditions were 94°C for 3 min for 1 cycle and then 94°C for 30 s, 54°C for 30 s, 72°C for 2 min for 38 cycles, 72°C for 7 min for 1 cycle.

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: 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). .. PCR was performed with thermal conditions as: 95°C for 10 min, 45 cycles of 95°C for 30 s, 57°C for 30 s and 72°C for 30 with a final extension of 72°C for 10 min.

Article Title: Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer
Article Snippet: Total RNA (20-50 μg) was added to 60 pmol primer of Oligo (dt) and heated at 65 °C for 10 min. .. 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: 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.

Article Title: The effect of centromere protein U silencing by lentiviral mediated RNA interference on the proliferation and apoptosis of breast cancer
Article Snippet: Paragraph title: RNA isolation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) ... Then, a RT reaction system, including 5×RT buffer (4.0 µl), RNA-free H2 O (2.6 µl), 10 mM dNTPs (2.0 µl; Promega, USA), RNasin (0.4 µl) and M-MLV-RTase (1.0 µl), was run for 1 h at 42°C, then transferred to a 70°C water bath for 10 min to inactivate the RTase.

Article Title: Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted anti-viral functions of APOBEC3G
Article Snippet: The HIV-1 RNA template was created using a dsDNA template for in vitro transcription that had been created by PCR using pNL4.3 as the template and the following primers: T7 HIV-1 TAR fwd 5’-aatttaatacgactcactataggggtctctctggttagaccag-3’ and UL244 rev 5'-gtcctgcgtcgagagatct-3' , . ssRNA was synthesized using the MAXIScript T7 transcription kit (Ambion) according to the manufacturer’s protocol, passed through a G-50 column and further purified by denaturing PAGE. .. Reactions of 10 μl were prepared in this buffer with 10 nM template, 200 μM dNTPs and 1 U/μl RNAase Inhibitor (Promega).

Article Title: Biofilm Formation by ica-Negative Ocular Isolates of Staphylococcus haemolyticus
Article Snippet: The extracted eDNA was diluted 10 times (1:9) using nuclease-free water and used as a template. .. PCR mixture contained: 4 μl of 5× PCR buffer, 1 μl of 25 mM MgCl2 , 2 μl each of dNTPs (Promega, Madison, WI, United States), 2 μl each of forward and reverse primer (5 pmol/μl) (GCC Biotech, New Delhi), 0.25 μl of 5 U/μl Taq polymerase (Promega, Madison, WI, United States), 1 μl of diluted eDNA, and 7.75 μl of nuclease-free water. .. The PCR was programmed as follows: initial denaturation at 94°C for 2 min, followed by 25 cycles consisting of denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 5 min. PCR products were electrophoresed on 1.5% agarose gel, stained with EtBr and photographed using a gel documentation system (Bio-Rad, United States).

Binding Assay:

Article Title: Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted anti-viral functions of APOBEC3G
Article Snippet: An 18 nt DNA oligonucleotide corresponding to the natural HIV-1 primer binding site (5'-gtccctgttcgggcgcca-3') was radiolabeled using polynucleotide kinase (NEB) and γ-P32 -ATP (Perkin Elmer). .. Reactions of 10 μl were prepared in this buffer with 10 nM template, 200 μM dNTPs and 1 U/μl RNAase Inhibitor (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: 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.

Article Title: Analysis of Zinc-Exporters Expression in Prostate Cancer
Article Snippet: The quality and quantity of isolated RNA were assessed using Synergy H1 hybrid multi-mode microplate reader (BioTek). .. First strand cDNA was transcribed with random primers, dNTPs and M-MLV reverse transcriptase (Promega).

Article Title: The effect of centromere protein U silencing by lentiviral mediated RNA interference on the proliferation and apoptosis of breast cancer
Article Snippet: Paragraph title: RNA isolation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) ... Then, a RT reaction system, including 5×RT buffer (4.0 µl), RNA-free H2 O (2.6 µl), 10 mM dNTPs (2.0 µl; Promega, USA), RNasin (0.4 µl) and M-MLV-RTase (1.0 µl), was run for 1 h at 42°C, then transferred to a 70°C water bath for 10 min to inactivate the RTase.

Labeling:

Article Title: Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted anti-viral functions of APOBEC3G
Article Snippet: This labeled primer and RNA template were annealed in 50 mM Tris HCl pH 8.0, 75 mM KCl at 65 °C for 5 min followed by gradual cooling to 37 °C, and annealed template/primer then diluted to 0.1 μM in reaction buffer (50 mM Tris HCl pH 8.0, 75 mM KCl, 7 mM MgCl2 , 1 mM DTT. .. Reactions of 10 μl were prepared in this buffer with 10 nM template, 200 μM dNTPs and 1 U/μl RNAase Inhibitor (Promega).

Purification:

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).

Article Title: Candidate genes associated with bud dormancy release in blackcurrant (Ribes nigrum L.)
Article Snippet: Each reaction was performed in 100 μl containing 50 ng of plasmid, M13 primers (0.5 μM each), 0.2 mM dNTPs, 2 mM dNTPs, 2 mM MgCl2 and 0.10 U/μl Taq DNA Polymerase (Promega) in 1 × PCR buffer. .. Each reaction was performed in 100 μl containing 50 ng of plasmid, M13 primers (0.5 μM each), 0.2 mM dNTPs, 2 mM dNTPs, 2 mM MgCl2 and 0.10 U/μl Taq DNA Polymerase (Promega) in 1 × PCR buffer.

Article Title: Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted anti-viral functions of APOBEC3G
Article Snippet: The HIV-1 RNA template was created using a dsDNA template for in vitro transcription that had been created by PCR using pNL4.3 as the template and the following primers: T7 HIV-1 TAR fwd 5’-aatttaatacgactcactataggggtctctctggttagaccag-3’ and UL244 rev 5'-gtcctgcgtcgagagatct-3' , . ssRNA was synthesized using the MAXIScript T7 transcription kit (Ambion) according to the manufacturer’s protocol, passed through a G-50 column and further purified by denaturing PAGE. .. Reactions of 10 μl were prepared in this buffer with 10 nM template, 200 μM dNTPs and 1 U/μl RNAase Inhibitor (Promega).

Reverse Transcription Polymerase Chain Reaction:

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[ , ].

Quantitative RT-PCR:

Article Title: Analysis of Zinc-Exporters Expression in Prostate Cancer
Article Snippet: Paragraph title: qRT-PCR analysis with human PCa tissue samples ... First strand cDNA was transcribed with random primers, dNTPs and M-MLV reverse transcriptase (Promega).

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.

Article Title: The effect of centromere protein U silencing by lentiviral mediated RNA interference on the proliferation and apoptosis of breast cancer
Article Snippet: Paragraph title: RNA isolation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) ... Then, a RT reaction system, including 5×RT buffer (4.0 µl), RNA-free H2 O (2.6 µl), 10 mM dNTPs (2.0 µl; Promega, USA), RNasin (0.4 µl) and M-MLV-RTase (1.0 µl), was run for 1 h at 42°C, then transferred to a 70°C water bath for 10 min to inactivate the RTase.

Polyacrylamide Gel Electrophoresis:

Article Title: Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted anti-viral functions of APOBEC3G
Article Snippet: The HIV-1 RNA template was created using a dsDNA template for in vitro transcription that had been created by PCR using pNL4.3 as the template and the following primers: T7 HIV-1 TAR fwd 5’-aatttaatacgactcactataggggtctctctggttagaccag-3’ and UL244 rev 5'-gtcctgcgtcgagagatct-3' , . ssRNA was synthesized using the MAXIScript T7 transcription kit (Ambion) according to the manufacturer’s protocol, passed through a G-50 column and further purified by denaturing PAGE. .. Reactions of 10 μl were prepared in this buffer with 10 nM template, 200 μM dNTPs and 1 U/μl RNAase Inhibitor (Promega).

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.

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.

Plasmid Preparation:

Article Title: Candidate genes associated with bud dormancy release in blackcurrant (Ribes nigrum L.)
Article Snippet: Blackcurrant cDNA inserts were amplified by PCR using plasmid DNA template and M13 forward and reverse primers that span the multiple cloning site of the vector. .. Each reaction was performed in 100 μl containing 50 ng of plasmid, M13 primers (0.5 μM each), 0.2 mM dNTPs, 2 mM dNTPs, 2 mM MgCl2 and 0.10 U/μl Taq DNA Polymerase (Promega) in 1 × PCR buffer. .. PCR conditions were 94°C for 3 min for 1 cycle and then 94°C for 30 s, 54°C for 30 s, 72°C for 2 min for 38 cycles, 72°C for 7 min for 1 cycle.

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). .. Primers were used at a final concentration of 250 nM along with 2 mM dNTPs (Promega, Hampshire, UK).

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). .. 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: Biofilm Formation by ica-Negative Ocular Isolates of Staphylococcus haemolyticus
Article Snippet: PCR mixture contained: 4 μl of 5× PCR buffer, 1 μl of 25 mM MgCl2 , 2 μl each of dNTPs (Promega, Madison, WI, United States), 2 μl each of forward and reverse primer (5 pmol/μl) (GCC Biotech, New Delhi), 0.25 μl of 5 U/μl Taq polymerase (Promega, Madison, WI, United States), 1 μl of diluted eDNA, and 7.75 μl of nuclease-free water. .. The PCR was programmed as follows: initial denaturation at 94°C for 2 min, followed by 25 cycles consisting of denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 5 min. PCR products were electrophoresed on 1.5% agarose gel, stained with EtBr and photographed using a gel documentation system (Bio-Rad, United States).

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). .. 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).

In Vitro:

Article Title: Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted anti-viral functions of APOBEC3G
Article Snippet: Paragraph title: In vitro primer extension assays ... Reactions of 10 μl were prepared in this buffer with 10 nM template, 200 μM dNTPs and 1 U/μl RNAase Inhibitor (Promega).

Spectrophotometry:

Article Title: The effect of centromere protein U silencing by lentiviral mediated RNA interference on the proliferation and apoptosis of breast cancer
Article Snippet: RNA purity and concentration were assessed using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Inc.). .. Then, a RT reaction system, including 5×RT buffer (4.0 µl), RNA-free H2 O (2.6 µl), 10 mM dNTPs (2.0 µl; Promega, USA), RNasin (0.4 µl) and M-MLV-RTase (1.0 µl), was run for 1 h at 42°C, then transferred to a 70°C water bath for 10 min to inactivate the RTase.

Concentration Assay:

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).

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: The reactions were performed using the PfuUltra II Fusion HS DNA polymerase (Agilent Technologies, UK). .. 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: Pre-treatment or Post-treatment of Human Glioma Cells With BIX01294, the Inhibitor of Histone Methyltransferase G9a, Sensitizes Cells to Temozolomide
Article Snippet: The purity and concentration of DNA was estimated at the absorbance of 260 and 280 nm. .. 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 effect of centromere protein U silencing by lentiviral mediated RNA interference on the proliferation and apoptosis of breast cancer
Article Snippet: RNA purity and concentration were assessed using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Inc.). .. Then, a RT reaction system, including 5×RT buffer (4.0 µl), RNA-free H2 O (2.6 µl), 10 mM dNTPs (2.0 µl; Promega, USA), RNasin (0.4 µl) and M-MLV-RTase (1.0 µl), was run for 1 h at 42°C, then transferred to a 70°C water bath for 10 min to inactivate the RTase.

Staining:

Article Title: Analysis of Zinc-Exporters Expression in Prostate Cancer
Article Snippet: Hematoxylin-eosin stained slides were marked according to non-hyperplastic benign areas and tumor areas. .. First strand cDNA was transcribed with random primers, dNTPs and M-MLV reverse transcriptase (Promega).

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    Promega dntps
    In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. <t>bRT</t> is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and <t>dNTPs,</t> including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.
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    In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.

    Article Snippet: Reactions were carried out in 20 μl containing 1.8 μM bRT-Avd, bRT or Avd, 100 ng/μl RNA template, 100 μM dNTPs (Promega) (or varying concentrations of certain dNTPs), 0.5 μCi/μl [α-32 P]dCTP, 20 units RNase inhibitor (NEB) in 75 mM KCl, 3 mM MgCl2 , 10 mM DTT, 50 mM HEPES, pH 7.5, 10% glycerol for 2 h at 37°C.

    Techniques: In Vitro, Binding Assay, Sequencing, Incubation, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Activity Assay, Produced

    Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Article Snippet: Reactions were carried out in 20 μl containing 1.8 μM bRT-Avd, bRT or Avd, 100 ng/μl RNA template, 100 μM dNTPs (Promega) (or varying concentrations of certain dNTPs), 0.5 μCi/μl [α-32 P]dCTP, 20 units RNase inhibitor (NEB) in 75 mM KCl, 3 mM MgCl2 , 10 mM DTT, 50 mM HEPES, pH 7.5, 10% glycerol for 2 h at 37°C.

    Techniques: Activity Assay, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Incubation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

    Article Snippet: Reactions were carried out in 20 μl containing 1.8 μM bRT-Avd, bRT or Avd, 100 ng/μl RNA template, 100 μM dNTPs (Promega) (or varying concentrations of certain dNTPs), 0.5 μCi/μl [α-32 P]dCTP, 20 units RNase inhibitor (NEB) in 75 mM KCl, 3 mM MgCl2 , 10 mM DTT, 50 mM HEPES, pH 7.5, 10% glycerol for 2 h at 37°C.

    Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Amplification, Produced, Sequencing, Activity Assay, Polyacrylamide Gel Electrophoresis

    Controlling the extent of pausing by AMV-RT at a 7-EAA site. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide. (B) Primer extension results for reactions containing 10 μM dNTPs at 42 °C for 45 min. Lanes are labeled as follows for N: −, labeled primer only, no extension; A, adenosine; 7, 7-EAA; B, biotin triazole; and B + S, biotin triazole + monomeric streptavidin. (C) Primer extension results for reactions containing 1 μM dNTPs at 37 °C for 5 min; lanes are labeled the same as in panel B, and the arrow indicates a pause site. (D) Quantification of inhibition of primer extension under the conditions used in panel C; the average for at least three independent primer extension reactions ± standard deviation is plotted.

    Journal: ACS Chemical Biology

    Article Title: Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA

    doi: 10.1021/cb500270x

    Figure Lengend Snippet: Controlling the extent of pausing by AMV-RT at a 7-EAA site. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide. (B) Primer extension results for reactions containing 10 μM dNTPs at 42 °C for 45 min. Lanes are labeled as follows for N: −, labeled primer only, no extension; A, adenosine; 7, 7-EAA; B, biotin triazole; and B + S, biotin triazole + monomeric streptavidin. (C) Primer extension results for reactions containing 1 μM dNTPs at 37 °C for 5 min; lanes are labeled the same as in panel B, and the arrow indicates a pause site. (D) Quantification of inhibition of primer extension under the conditions used in panel C; the average for at least three independent primer extension reactions ± standard deviation is plotted.

    Article Snippet: Samples were then mixed with AMV-RT and dNTP mix so that the final concentrations were as follows: 20 nM RNA, ∼80 nM 32 P-labeled GluR B pre-mRNA 18 nt primer, 10 μM dNTPs, 1× Promega AMV-RT buffer, and 5 units of AMV-RT for the standard extension conditions protocol and 20 nM RNA, ∼80 nM 32 P-labeled GluR B pre-mRNA 18 nt primer, 1 μM dNTPs, 1× Promega AMV-RT buffer, and 5 units of AMV-RT for the low [dNTP] conditions protocol.

    Techniques: Primer Extension Assay, Labeling, Inhibition, Standard Deviation