Structured Review

Roche t7 rna polymerase
Secondary structure presentation of ( A ) mature B. subtilis 6S-1 RNA (190 nt), adapted from ( 11 ), and ( B ) of E. coli 6S RNA according to ( 9 ); both RNAs were transcribed with two artificially added G residues (lowercase letters) for reasons of efficient synthesis by <t>T7</t> RNA polymerase. In both panels, the black lines along the sequence indicate the region of B. subtilis 6S-1 RNA and E. coli 6S RNA that serve as a template for the synthesis of small transcripts (product RNAs = pRNAs) by RNA polymerase (this study, 9); arrows mark the starting point of pRNA transcription; the chemically synthesized pRNA mimics are depicted in the grey boxes on the left above the 6S RNA structures. In panel A, 6S-1 RNA nt 151–157 complementary to nt 2–8 of the LNA probe are boxed. LNA probes for pRNA detection are shown in grey boxes on the right above the secondary structures; DIG, digoxigenin attached to the 5′-end via a C6 linker; small letters depict DNA residues, capital letters LNA residues.
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Images

1) Product Images from "Northern blot detection of endogenous small RNAs (~14 nt) in bacterial total RNA extracts"

Article Title: Northern blot detection of endogenous small RNAs (~14 nt) in bacterial total RNA extracts

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq437

Secondary structure presentation of ( A ) mature B. subtilis 6S-1 RNA (190 nt), adapted from ( 11 ), and ( B ) of E. coli 6S RNA according to ( 9 ); both RNAs were transcribed with two artificially added G residues (lowercase letters) for reasons of efficient synthesis by T7 RNA polymerase. In both panels, the black lines along the sequence indicate the region of B. subtilis 6S-1 RNA and E. coli 6S RNA that serve as a template for the synthesis of small transcripts (product RNAs = pRNAs) by RNA polymerase (this study, 9); arrows mark the starting point of pRNA transcription; the chemically synthesized pRNA mimics are depicted in the grey boxes on the left above the 6S RNA structures. In panel A, 6S-1 RNA nt 151–157 complementary to nt 2–8 of the LNA probe are boxed. LNA probes for pRNA detection are shown in grey boxes on the right above the secondary structures; DIG, digoxigenin attached to the 5′-end via a C6 linker; small letters depict DNA residues, capital letters LNA residues.
Figure Legend Snippet: Secondary structure presentation of ( A ) mature B. subtilis 6S-1 RNA (190 nt), adapted from ( 11 ), and ( B ) of E. coli 6S RNA according to ( 9 ); both RNAs were transcribed with two artificially added G residues (lowercase letters) for reasons of efficient synthesis by T7 RNA polymerase. In both panels, the black lines along the sequence indicate the region of B. subtilis 6S-1 RNA and E. coli 6S RNA that serve as a template for the synthesis of small transcripts (product RNAs = pRNAs) by RNA polymerase (this study, 9); arrows mark the starting point of pRNA transcription; the chemically synthesized pRNA mimics are depicted in the grey boxes on the left above the 6S RNA structures. In panel A, 6S-1 RNA nt 151–157 complementary to nt 2–8 of the LNA probe are boxed. LNA probes for pRNA detection are shown in grey boxes on the right above the secondary structures; DIG, digoxigenin attached to the 5′-end via a C6 linker; small letters depict DNA residues, capital letters LNA residues.

Techniques Used: Sequencing, Synthesized

2) Product Images from "Exploiting cis-Acting Replication Elements To Direct Hepatitis C Virus-Dependent Transgene Expression"

Article Title: Exploiting cis-Acting Replication Elements To Direct Hepatitis C Virus-Dependent Transgene Expression

Journal: Journal of Virology

doi: 10.1128/JVI.79.10.5923-5932.2005

Determination of cis -acting replication elements in the NS5B coding region. (A) Schematic display of T7-based HCV minigenome reporter constructs. HCV minigenome containing the antisense (left) or sense (right) sequence of the Renilla luciferase gene and the antisense sequence of the EMCV IRES flanked by the 5′ end (1 to 377) and differently truncated NS5B coding region-connected 3′ UTR or 3′ UTR alone was juxtaposed precisely at the T7 transcription start site and followed by the HDV ribozyme gene. (B) The indicated reporter vectors were transfected into Huh-NNRZ cells with or without pAM8-1 expressing T7 RNA polymerase. Relative Renilla luciferase activities in the lysates were determined at 48 h posttransfection. The columns and bars represent the means and standard deviations of three independent triplicate transfections. (C) Huh-NNRZ cells were transfected with in vitro transcribed cRLNS5B1 RNA (left) or EMCVRLNS5B1 RNA (right) together with capped firefly luciferase RNA as an internal control. Relative Renilla luciferase activities in the lysates were determined at 24 h posttransfection. Absolute values of Renilla and firefly luciferase activity are listed below the corresponding bars.
Figure Legend Snippet: Determination of cis -acting replication elements in the NS5B coding region. (A) Schematic display of T7-based HCV minigenome reporter constructs. HCV minigenome containing the antisense (left) or sense (right) sequence of the Renilla luciferase gene and the antisense sequence of the EMCV IRES flanked by the 5′ end (1 to 377) and differently truncated NS5B coding region-connected 3′ UTR or 3′ UTR alone was juxtaposed precisely at the T7 transcription start site and followed by the HDV ribozyme gene. (B) The indicated reporter vectors were transfected into Huh-NNRZ cells with or without pAM8-1 expressing T7 RNA polymerase. Relative Renilla luciferase activities in the lysates were determined at 48 h posttransfection. The columns and bars represent the means and standard deviations of three independent triplicate transfections. (C) Huh-NNRZ cells were transfected with in vitro transcribed cRLNS5B1 RNA (left) or EMCVRLNS5B1 RNA (right) together with capped firefly luciferase RNA as an internal control. Relative Renilla luciferase activities in the lysates were determined at 24 h posttransfection. Absolute values of Renilla and firefly luciferase activity are listed below the corresponding bars.

Techniques Used: Construct, Sequencing, Luciferase, Transfection, Expressing, In Vitro, Activity Assay

3) Product Images from "Human mitochondrial RNA polymerase primes lagging-strand DNA synthesis in vitro"

Article Title: Human mitochondrial RNA polymerase primes lagging-strand DNA synthesis in vitro

Journal:

doi: 10.1073/pnas.0805399105

RNA synthesis of POLRMT and T7 RNA polymerase on ssDNA and dsDNA templates. RNA synthesis by POLRMT (500 fmol) or by T7 RNA polymerase (0.8 units) were monitored as described in Material and Methods . ( A ) RNA products formed on the M13mp18 ssDNA template
Figure Legend Snippet: RNA synthesis of POLRMT and T7 RNA polymerase on ssDNA and dsDNA templates. RNA synthesis by POLRMT (500 fmol) or by T7 RNA polymerase (0.8 units) were monitored as described in Material and Methods . ( A ) RNA products formed on the M13mp18 ssDNA template

Techniques Used:

4) Product Images from "R-Loop Formation In Trans at an AGGAG Repeat"

Article Title: R-Loop Formation In Trans at an AGGAG Repeat

Journal: Journal of Nucleic Acids

doi: 10.1155/2013/629218

R-loop formation in trans detected by radiolabeling of the transcript. (a) Agarose gel stained with ethidium bromide and (b) autoradiogram of the same gel. Lane 1: pBluescript SK(−) linearized with Xba I; lane 2: supercoiled pHC624-(AGGAG) 22 ; lane 3: pHC624-(AGGAG) 22 linearized with Sca I; lane 4: pSK-(AGGAG) 22 linearized with Xba I; lane 5: linearized pSK-(AGGAG) 22 and supercoiled pHC624-(AGGAG) 22 ; and lane 6: linearized pSK-(AGGAG) 22 and linearized pHC624-(AGGAG) 22 . All DNAs were incubated in the transcription mixture with T7 RNA polymerase.
Figure Legend Snippet: R-loop formation in trans detected by radiolabeling of the transcript. (a) Agarose gel stained with ethidium bromide and (b) autoradiogram of the same gel. Lane 1: pBluescript SK(−) linearized with Xba I; lane 2: supercoiled pHC624-(AGGAG) 22 ; lane 3: pHC624-(AGGAG) 22 linearized with Sca I; lane 4: pSK-(AGGAG) 22 linearized with Xba I; lane 5: linearized pSK-(AGGAG) 22 and supercoiled pHC624-(AGGAG) 22 ; and lane 6: linearized pSK-(AGGAG) 22 and linearized pHC624-(AGGAG) 22 . All DNAs were incubated in the transcription mixture with T7 RNA polymerase.

Techniques Used: Radioactivity, Agarose Gel Electrophoresis, Staining, Incubation

R-loop formation on pSK-(AGGAG) 22 and effects of RNase T1 on the formation. A plasmid containing a (AGGAG) 22 repeat was transcribed with T7 RNA polymerase and the effects on the topology of the plasmid were examined by agarose gel electrophoresis. (a) Lane 1: mock transcribed; lane 2: transcribed with T7 RNA polymerase; lane 3: transcribed with T3 RNA polymerase. (b) lane 1: mock transcribed, lane 2: transcribed with T7 RNA polymerase, Lane 3: transcribed with T7 RNA polymerase in the presence of 3 units of RNase T1; lane 4: transcribed with T7 RNA polymerase and ethanol-precipitated; and lane 5: transcribed with T7 RNA polymerase, ethanol-precipitated, and then incubated with 3 units of RNase T1.
Figure Legend Snippet: R-loop formation on pSK-(AGGAG) 22 and effects of RNase T1 on the formation. A plasmid containing a (AGGAG) 22 repeat was transcribed with T7 RNA polymerase and the effects on the topology of the plasmid were examined by agarose gel electrophoresis. (a) Lane 1: mock transcribed; lane 2: transcribed with T7 RNA polymerase; lane 3: transcribed with T3 RNA polymerase. (b) lane 1: mock transcribed, lane 2: transcribed with T7 RNA polymerase, Lane 3: transcribed with T7 RNA polymerase in the presence of 3 units of RNase T1; lane 4: transcribed with T7 RNA polymerase and ethanol-precipitated; and lane 5: transcribed with T7 RNA polymerase, ethanol-precipitated, and then incubated with 3 units of RNase T1.

Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Incubation

R-loop formation in trans and its dependence on supercoiling of the target DNA. RNA containing an AGGAG repeat was produced from linearized pSK-(AGGAG) 22 , and the effects on a T7 promoterless plasmid containing an AGGAG repeat, pHC624-(AGGAG) 22 , were examined by agarose gel electrophoresis. Lane 1: pSK-(AGGAG) 22 linearized with Xba I; lane 2: linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 3: supercoiled pHC624-(AGGAG) 22 , which lacked T7 promoter; lane 4: supercoiled pHC624-(AGGAG) 22 incubated in transcription mixture with T7 RNA polymerase; lane 5: supercoiled pHC624-(AGGAG) 22 and linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 6: supercoiled pHC624-(AGGAG) 22 and pBluescript SK(−) linearized with Xba I, transcribed with T7 RNA polymerase; lane 7: supercoiled pHC624; lane 8: supercoiled pHC624 and linearized pSK-(AGGAG) 22 incubated in transcription mixture with T7 RNA polymerase; lane 9: pHC624-(AGGAG) 22 relaxed with vaccinia topoisomerase I; lane 10: relaxed pHC624-(AGAAG) 22 and linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 11: same as lane 5 and ethanol-precipitated; lane 12: same as lane 11 and treated with 90 units of E. coli RNase H (TaKaRa) in 40 mM Tris · HCl pH 7.7, 4 mM MgCl 2 , 1 mM DTT, 4% glycerol, and 0.003% bovine serum albumin at 37°C for 1 h.
Figure Legend Snippet: R-loop formation in trans and its dependence on supercoiling of the target DNA. RNA containing an AGGAG repeat was produced from linearized pSK-(AGGAG) 22 , and the effects on a T7 promoterless plasmid containing an AGGAG repeat, pHC624-(AGGAG) 22 , were examined by agarose gel electrophoresis. Lane 1: pSK-(AGGAG) 22 linearized with Xba I; lane 2: linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 3: supercoiled pHC624-(AGGAG) 22 , which lacked T7 promoter; lane 4: supercoiled pHC624-(AGGAG) 22 incubated in transcription mixture with T7 RNA polymerase; lane 5: supercoiled pHC624-(AGGAG) 22 and linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 6: supercoiled pHC624-(AGGAG) 22 and pBluescript SK(−) linearized with Xba I, transcribed with T7 RNA polymerase; lane 7: supercoiled pHC624; lane 8: supercoiled pHC624 and linearized pSK-(AGGAG) 22 incubated in transcription mixture with T7 RNA polymerase; lane 9: pHC624-(AGGAG) 22 relaxed with vaccinia topoisomerase I; lane 10: relaxed pHC624-(AGAAG) 22 and linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 11: same as lane 5 and ethanol-precipitated; lane 12: same as lane 11 and treated with 90 units of E. coli RNase H (TaKaRa) in 40 mM Tris · HCl pH 7.7, 4 mM MgCl 2 , 1 mM DTT, 4% glycerol, and 0.003% bovine serum albumin at 37°C for 1 h.

Techniques Used: Produced, Plasmid Preparation, Agarose Gel Electrophoresis, Incubation

5) Product Images from "Comprehensive Secondary Structure Elucidation of Four Genera of the Family Pospiviroidae"

Article Title: Comprehensive Secondary Structure Elucidation of Four Genera of the Family Pospiviroidae

Journal: PLoS ONE

doi: 10.1371/journal.pone.0098655

Schematic flow chart illustrating the detailed steps of the hSHAPE experiment with dimeric PSTVd. The numbered primers are used for the amplification of the PSTVd monomers (i.e. 1F/1R and 2F/2R). The raised ends of the primers represent the non-complementary regions, specifically the T7 RNA polymerase promoter, which is used for the run-off transcription. The RNAs obtained after amplification and transcription are numbered 1 and 2. These RNAs were subjected to the SHAPE reaction followed by primer extension. During the latter step the reverse transcriptase produces a cDNA fragment (dashed line) the length of the distance from the start of the RNA until the first adduct (e.g. circle on top of a line).
Figure Legend Snippet: Schematic flow chart illustrating the detailed steps of the hSHAPE experiment with dimeric PSTVd. The numbered primers are used for the amplification of the PSTVd monomers (i.e. 1F/1R and 2F/2R). The raised ends of the primers represent the non-complementary regions, specifically the T7 RNA polymerase promoter, which is used for the run-off transcription. The RNAs obtained after amplification and transcription are numbered 1 and 2. These RNAs were subjected to the SHAPE reaction followed by primer extension. During the latter step the reverse transcriptase produces a cDNA fragment (dashed line) the length of the distance from the start of the RNA until the first adduct (e.g. circle on top of a line).

Techniques Used: Flow Cytometry, Amplification

6) Product Images from "R-Loop Formation In Trans at an AGGAG Repeat"

Article Title: R-Loop Formation In Trans at an AGGAG Repeat

Journal: Journal of Nucleic Acids

doi: 10.1155/2013/629218

R-loop formation in trans detected by radiolabeling of the transcript. (a) Agarose gel stained with ethidium bromide and (b) autoradiogram of the same gel. Lane 1: pBluescript SK(−) linearized with Xba I; lane 2: supercoiled pHC624-(AGGAG) 22 ; lane 3: pHC624-(AGGAG) 22 linearized with Sca I; lane 4: pSK-(AGGAG) 22 linearized with Xba I; lane 5: linearized pSK-(AGGAG) 22 and supercoiled pHC624-(AGGAG) 22 ; and lane 6: linearized pSK-(AGGAG) 22 and linearized pHC624-(AGGAG) 22 . All DNAs were incubated in the transcription mixture with T7 RNA polymerase.
Figure Legend Snippet: R-loop formation in trans detected by radiolabeling of the transcript. (a) Agarose gel stained with ethidium bromide and (b) autoradiogram of the same gel. Lane 1: pBluescript SK(−) linearized with Xba I; lane 2: supercoiled pHC624-(AGGAG) 22 ; lane 3: pHC624-(AGGAG) 22 linearized with Sca I; lane 4: pSK-(AGGAG) 22 linearized with Xba I; lane 5: linearized pSK-(AGGAG) 22 and supercoiled pHC624-(AGGAG) 22 ; and lane 6: linearized pSK-(AGGAG) 22 and linearized pHC624-(AGGAG) 22 . All DNAs were incubated in the transcription mixture with T7 RNA polymerase.

Techniques Used: Radioactivity, Agarose Gel Electrophoresis, Staining, Incubation

R-loop formation on pSK-(AGGAG) 22 and effects of RNase T1 on the formation. A plasmid containing a (AGGAG) 22 repeat was transcribed with T7 RNA polymerase and the effects on the topology of the plasmid were examined by agarose gel electrophoresis. (a) Lane 1: mock transcribed; lane 2: transcribed with T7 RNA polymerase; lane 3: transcribed with T3 RNA polymerase. (b) lane 1: mock transcribed, lane 2: transcribed with T7 RNA polymerase, Lane 3: transcribed with T7 RNA polymerase in the presence of 3 units of RNase T1; lane 4: transcribed with T7 RNA polymerase and ethanol-precipitated; and lane 5: transcribed with T7 RNA polymerase, ethanol-precipitated, and then incubated with 3 units of RNase T1.
Figure Legend Snippet: R-loop formation on pSK-(AGGAG) 22 and effects of RNase T1 on the formation. A plasmid containing a (AGGAG) 22 repeat was transcribed with T7 RNA polymerase and the effects on the topology of the plasmid were examined by agarose gel electrophoresis. (a) Lane 1: mock transcribed; lane 2: transcribed with T7 RNA polymerase; lane 3: transcribed with T3 RNA polymerase. (b) lane 1: mock transcribed, lane 2: transcribed with T7 RNA polymerase, Lane 3: transcribed with T7 RNA polymerase in the presence of 3 units of RNase T1; lane 4: transcribed with T7 RNA polymerase and ethanol-precipitated; and lane 5: transcribed with T7 RNA polymerase, ethanol-precipitated, and then incubated with 3 units of RNase T1.

Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Incubation

R-loop formation in trans and its dependence on supercoiling of the target DNA. RNA containing an AGGAG repeat was produced from linearized pSK-(AGGAG) 22 , and the effects on a T7 promoterless plasmid containing an AGGAG repeat, pHC624-(AGGAG) 22 , were examined by agarose gel electrophoresis. Lane 1: pSK-(AGGAG) 22 linearized with Xba I; lane 2: linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 3: supercoiled pHC624-(AGGAG) 22 , which lacked T7 promoter; lane 4: supercoiled pHC624-(AGGAG) 22 incubated in transcription mixture with T7 RNA polymerase; lane 5: supercoiled pHC624-(AGGAG) 22 and linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 6: supercoiled pHC624-(AGGAG) 22 and pBluescript SK(−) linearized with Xba I, transcribed with T7 RNA polymerase; lane 7: supercoiled pHC624; lane 8: supercoiled pHC624 and linearized pSK-(AGGAG) 22 incubated in transcription mixture with T7 RNA polymerase; lane 9: pHC624-(AGGAG) 22 relaxed with vaccinia topoisomerase I; lane 10: relaxed pHC624-(AGAAG) 22 and linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 11: same as lane 5 and ethanol-precipitated; lane 12: same as lane 11 and treated with 90 units of E. coli RNase H (TaKaRa) in 40 mM Tris · HCl pH 7.7, 4 mM MgCl 2 , 1 mM DTT, 4% glycerol, and 0.003% bovine serum albumin at 37°C for 1 h.
Figure Legend Snippet: R-loop formation in trans and its dependence on supercoiling of the target DNA. RNA containing an AGGAG repeat was produced from linearized pSK-(AGGAG) 22 , and the effects on a T7 promoterless plasmid containing an AGGAG repeat, pHC624-(AGGAG) 22 , were examined by agarose gel electrophoresis. Lane 1: pSK-(AGGAG) 22 linearized with Xba I; lane 2: linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 3: supercoiled pHC624-(AGGAG) 22 , which lacked T7 promoter; lane 4: supercoiled pHC624-(AGGAG) 22 incubated in transcription mixture with T7 RNA polymerase; lane 5: supercoiled pHC624-(AGGAG) 22 and linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 6: supercoiled pHC624-(AGGAG) 22 and pBluescript SK(−) linearized with Xba I, transcribed with T7 RNA polymerase; lane 7: supercoiled pHC624; lane 8: supercoiled pHC624 and linearized pSK-(AGGAG) 22 incubated in transcription mixture with T7 RNA polymerase; lane 9: pHC624-(AGGAG) 22 relaxed with vaccinia topoisomerase I; lane 10: relaxed pHC624-(AGAAG) 22 and linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 11: same as lane 5 and ethanol-precipitated; lane 12: same as lane 11 and treated with 90 units of E. coli RNase H (TaKaRa) in 40 mM Tris · HCl pH 7.7, 4 mM MgCl 2 , 1 mM DTT, 4% glycerol, and 0.003% bovine serum albumin at 37°C for 1 h.

Techniques Used: Produced, Plasmid Preparation, Agarose Gel Electrophoresis, Incubation

7) Product Images from "R-Loop Formation In Trans at an AGGAG Repeat"

Article Title: R-Loop Formation In Trans at an AGGAG Repeat

Journal: Journal of Nucleic Acids

doi: 10.1155/2013/629218

R-loop formation in trans detected by radiolabeling of the transcript. (a) Agarose gel stained with ethidium bromide and (b) autoradiogram of the same gel. Lane 1: pBluescript SK(−) linearized with Xba I; lane 2: supercoiled pHC624-(AGGAG) 22 ; lane 3: pHC624-(AGGAG) 22 linearized with Sca I; lane 4: pSK-(AGGAG) 22 linearized with Xba I; lane 5: linearized pSK-(AGGAG) 22 and supercoiled pHC624-(AGGAG) 22 ; and lane 6: linearized pSK-(AGGAG) 22 and linearized pHC624-(AGGAG) 22 . All DNAs were incubated in the transcription mixture with T7 RNA polymerase.
Figure Legend Snippet: R-loop formation in trans detected by radiolabeling of the transcript. (a) Agarose gel stained with ethidium bromide and (b) autoradiogram of the same gel. Lane 1: pBluescript SK(−) linearized with Xba I; lane 2: supercoiled pHC624-(AGGAG) 22 ; lane 3: pHC624-(AGGAG) 22 linearized with Sca I; lane 4: pSK-(AGGAG) 22 linearized with Xba I; lane 5: linearized pSK-(AGGAG) 22 and supercoiled pHC624-(AGGAG) 22 ; and lane 6: linearized pSK-(AGGAG) 22 and linearized pHC624-(AGGAG) 22 . All DNAs were incubated in the transcription mixture with T7 RNA polymerase.

Techniques Used: Radioactivity, Agarose Gel Electrophoresis, Staining, Incubation

R-loop formation on pSK-(AGGAG) 22 and effects of RNase T1 on the formation. A plasmid containing a (AGGAG) 22 repeat was transcribed with T7 RNA polymerase and the effects on the topology of the plasmid were examined by agarose gel electrophoresis. (a) Lane 1: mock transcribed; lane 2: transcribed with T7 RNA polymerase; lane 3: transcribed with T3 RNA polymerase. (b) lane 1: mock transcribed, lane 2: transcribed with T7 RNA polymerase, Lane 3: transcribed with T7 RNA polymerase in the presence of 3 units of RNase T1; lane 4: transcribed with T7 RNA polymerase and ethanol-precipitated; and lane 5: transcribed with T7 RNA polymerase, ethanol-precipitated, and then incubated with 3 units of RNase T1.
Figure Legend Snippet: R-loop formation on pSK-(AGGAG) 22 and effects of RNase T1 on the formation. A plasmid containing a (AGGAG) 22 repeat was transcribed with T7 RNA polymerase and the effects on the topology of the plasmid were examined by agarose gel electrophoresis. (a) Lane 1: mock transcribed; lane 2: transcribed with T7 RNA polymerase; lane 3: transcribed with T3 RNA polymerase. (b) lane 1: mock transcribed, lane 2: transcribed with T7 RNA polymerase, Lane 3: transcribed with T7 RNA polymerase in the presence of 3 units of RNase T1; lane 4: transcribed with T7 RNA polymerase and ethanol-precipitated; and lane 5: transcribed with T7 RNA polymerase, ethanol-precipitated, and then incubated with 3 units of RNase T1.

Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Incubation

R-loop formation in trans and its dependence on supercoiling of the target DNA. RNA containing an AGGAG repeat was produced from linearized pSK-(AGGAG) 22 , and the effects on a T7 promoterless plasmid containing an AGGAG repeat, pHC624-(AGGAG) 22 , were examined by agarose gel electrophoresis. Lane 1: pSK-(AGGAG) 22 linearized with Xba I; lane 2: linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 3: supercoiled pHC624-(AGGAG) 22 , which lacked T7 promoter; lane 4: supercoiled pHC624-(AGGAG) 22 incubated in transcription mixture with T7 RNA polymerase; lane 5: supercoiled pHC624-(AGGAG) 22 and linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 6: supercoiled pHC624-(AGGAG) 22 and pBluescript SK(−) linearized with Xba I, transcribed with T7 RNA polymerase; lane 7: supercoiled pHC624; lane 8: supercoiled pHC624 and linearized pSK-(AGGAG) 22 incubated in transcription mixture with T7 RNA polymerase; lane 9: pHC624-(AGGAG) 22 relaxed with vaccinia topoisomerase I; lane 10: relaxed pHC624-(AGAAG) 22 and linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 11: same as lane 5 and ethanol-precipitated; lane 12: same as lane 11 and treated with 90 units of E. coli RNase H (TaKaRa) in 40 mM Tris · HCl pH 7.7, 4 mM MgCl 2 , 1 mM DTT, 4% glycerol, and 0.003% bovine serum albumin at 37°C for 1 h.
Figure Legend Snippet: R-loop formation in trans and its dependence on supercoiling of the target DNA. RNA containing an AGGAG repeat was produced from linearized pSK-(AGGAG) 22 , and the effects on a T7 promoterless plasmid containing an AGGAG repeat, pHC624-(AGGAG) 22 , were examined by agarose gel electrophoresis. Lane 1: pSK-(AGGAG) 22 linearized with Xba I; lane 2: linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 3: supercoiled pHC624-(AGGAG) 22 , which lacked T7 promoter; lane 4: supercoiled pHC624-(AGGAG) 22 incubated in transcription mixture with T7 RNA polymerase; lane 5: supercoiled pHC624-(AGGAG) 22 and linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 6: supercoiled pHC624-(AGGAG) 22 and pBluescript SK(−) linearized with Xba I, transcribed with T7 RNA polymerase; lane 7: supercoiled pHC624; lane 8: supercoiled pHC624 and linearized pSK-(AGGAG) 22 incubated in transcription mixture with T7 RNA polymerase; lane 9: pHC624-(AGGAG) 22 relaxed with vaccinia topoisomerase I; lane 10: relaxed pHC624-(AGAAG) 22 and linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 11: same as lane 5 and ethanol-precipitated; lane 12: same as lane 11 and treated with 90 units of E. coli RNase H (TaKaRa) in 40 mM Tris · HCl pH 7.7, 4 mM MgCl 2 , 1 mM DTT, 4% glycerol, and 0.003% bovine serum albumin at 37°C for 1 h.

Techniques Used: Produced, Plasmid Preparation, Agarose Gel Electrophoresis, Incubation

8) Product Images from "Comprehensive Secondary Structure Elucidation of Four Genera of the Family Pospiviroidae"

Article Title: Comprehensive Secondary Structure Elucidation of Four Genera of the Family Pospiviroidae

Journal: PLoS ONE

doi: 10.1371/journal.pone.0098655

Schematic flow chart illustrating the detailed steps of the hSHAPE experiment with dimeric PSTVd. The numbered primers are used for the amplification of the PSTVd monomers (i.e. 1F/1R and 2F/2R). The raised ends of the primers represent the non-complementary regions, specifically the T7 RNA polymerase promoter, which is used for the run-off transcription. The RNAs obtained after amplification and transcription are numbered 1 and 2. These RNAs were subjected to the SHAPE reaction followed by primer extension. During the latter step the reverse transcriptase produces a cDNA fragment (dashed line) the length of the distance from the start of the RNA until the first adduct (e.g. circle on top of a line).
Figure Legend Snippet: Schematic flow chart illustrating the detailed steps of the hSHAPE experiment with dimeric PSTVd. The numbered primers are used for the amplification of the PSTVd monomers (i.e. 1F/1R and 2F/2R). The raised ends of the primers represent the non-complementary regions, specifically the T7 RNA polymerase promoter, which is used for the run-off transcription. The RNAs obtained after amplification and transcription are numbered 1 and 2. These RNAs were subjected to the SHAPE reaction followed by primer extension. During the latter step the reverse transcriptase produces a cDNA fragment (dashed line) the length of the distance from the start of the RNA until the first adduct (e.g. circle on top of a line).

Techniques Used: Flow Cytometry, Amplification

9) Product Images from "A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell‐free protein synthesis systems"

Article Title: A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell‐free protein synthesis systems

Journal: Biotechnology Progress

doi: 10.1002/btpr.2082

The costs of cell‐free reagents plotted according to their category in Table 1. Cell‐free S30 extract, T7 RNA polymerase and DNA template can be readily produced in‐house and have been excluded from the cost analysis.
Figure Legend Snippet: The costs of cell‐free reagents plotted according to their category in Table 1. Cell‐free S30 extract, T7 RNA polymerase and DNA template can be readily produced in‐house and have been excluded from the cost analysis.

Techniques Used: Produced

10) Product Images from "Adenosylcobalamin inhibits ribosome binding to btuB RNA"

Article Title: Adenosylcobalamin inhibits ribosome binding to btuB RNA

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

doi:

( A Upper ) Schematic structure of the btuB regulatory region. The positions of regulatory elements are indicated relative to the transcription start site and the start of the btuB coding sequence. The region from +1 to +315 is carried on the DNA template for synthesis of btuB RNA, with transcription from the T7 φ10 late promoter; the region for primer hybridization in the toe-print assay is indicated ( Right ). ( Lower ) The major primer extension products are identified by the nucleotide position of their 3′ end. ( B Lower ) Ribosome binding to lac and btuB RNA. RNA containing the translation initiation region from lacZ and btuB genes were synthesized by using T7 RNA polymerase and used in the primer extension inhibition assay. Incubation in the presence of tRNA fMet , 20 nM 30S ribosomal subunits, or 5 μM Ado-Cbl, as indicated ( Upper ), was for 10 min; primer extension was then carried out for 10 min after addition of AMV reverse transcriptase. The four lanes ( Left ) (A,C,G,T) are sequencing ladders for the btuB region; ( Righ t) the positions of the last base of the primer extension fragments.
Figure Legend Snippet: ( A Upper ) Schematic structure of the btuB regulatory region. The positions of regulatory elements are indicated relative to the transcription start site and the start of the btuB coding sequence. The region from +1 to +315 is carried on the DNA template for synthesis of btuB RNA, with transcription from the T7 φ10 late promoter; the region for primer hybridization in the toe-print assay is indicated ( Right ). ( Lower ) The major primer extension products are identified by the nucleotide position of their 3′ end. ( B Lower ) Ribosome binding to lac and btuB RNA. RNA containing the translation initiation region from lacZ and btuB genes were synthesized by using T7 RNA polymerase and used in the primer extension inhibition assay. Incubation in the presence of tRNA fMet , 20 nM 30S ribosomal subunits, or 5 μM Ado-Cbl, as indicated ( Upper ), was for 10 min; primer extension was then carried out for 10 min after addition of AMV reverse transcriptase. The four lanes ( Left ) (A,C,G,T) are sequencing ladders for the btuB region; ( Righ t) the positions of the last base of the primer extension fragments.

Techniques Used: Sequencing, Hybridization, Binding Assay, Synthesized, Toeprinting Assay, Incubation

11) Product Images from "Identification of cellular proteins enhancing activities of internal ribosomal entry sites by competition with oligodeoxynucleotides"

Article Title: Identification of cellular proteins enhancing activities of internal ribosomal entry sites by competition with oligodeoxynucleotides

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkh300

Effect of the CT-oligomer on IRES-dependent translation. ( A ) A schematic diagram of the reporter constructs. Capped mRNAs were generated by in vitro transcription with T7 RNA polymerase in the presence of m 7 GpppG. The mRNAs (RPF, RHF and RRF) contain
Figure Legend Snippet: Effect of the CT-oligomer on IRES-dependent translation. ( A ) A schematic diagram of the reporter constructs. Capped mRNAs were generated by in vitro transcription with T7 RNA polymerase in the presence of m 7 GpppG. The mRNAs (RPF, RHF and RRF) contain

Techniques Used: Construct, Generated, In Vitro

12) Product Images from "The Cloning and Characterization of the Enolase2 Gene of Gekko japonicus and Its Polyclonal Antibody Preparation"

Article Title: The Cloning and Characterization of the Enolase2 Gene of Gekko japonicus and Its Polyclonal Antibody Preparation

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms14058787

NSE mRNA transcript in CNS, the expression pattern in different tissues and mRNA expression after spinal cord transection in geckos. ( A ) The representative result of Northern blotting analyses of NSE mRNA in CNS of adult gecko. Ten micrograms of total RNA samples from brain and spinal cord of gecko were separated in 1% formaldehyde-denatured ( w / v ) agarose gel ( Left panel ), with the 18S and 28S rRNAs of corresponding tissues shown on the left. The 680 bp from NSE-open read frame (ORF) sequence was obtained by the PCR method and cloned into the pGEM-T Easy vector. Dig-labeled NSE riboprobes were synthesized in vitro from linearized plasmid above with T7 RNA polymerase. The hybridized bands were visualized with CDP-Star chemiluminescent substrate and recorded by X-ray film. The result of Northern blotting ( Right panel ) showed the length of NSE mRNA about 2.2 kb; ( B ) the representative results of semi-quantitative RT-PCR analyses of NSE mRNA in the different tissues of adult geckos, including liver, lung, kidney, heart, spinal cord, brain and ovary of adult geckos. The results revealed that the expression level of NSE mRNA was high in brain, spinal cord and low in heart, while it was not detectable in other tissues; ( C ) real-time qPCR analysis of NSE expression in the spinal cord after transection for the controls (Ctrl) and one, three and seven days post-injury. EF-1α was used for the quantitative normalization. * 1 d vs . Ctrl, p
Figure Legend Snippet: NSE mRNA transcript in CNS, the expression pattern in different tissues and mRNA expression after spinal cord transection in geckos. ( A ) The representative result of Northern blotting analyses of NSE mRNA in CNS of adult gecko. Ten micrograms of total RNA samples from brain and spinal cord of gecko were separated in 1% formaldehyde-denatured ( w / v ) agarose gel ( Left panel ), with the 18S and 28S rRNAs of corresponding tissues shown on the left. The 680 bp from NSE-open read frame (ORF) sequence was obtained by the PCR method and cloned into the pGEM-T Easy vector. Dig-labeled NSE riboprobes were synthesized in vitro from linearized plasmid above with T7 RNA polymerase. The hybridized bands were visualized with CDP-Star chemiluminescent substrate and recorded by X-ray film. The result of Northern blotting ( Right panel ) showed the length of NSE mRNA about 2.2 kb; ( B ) the representative results of semi-quantitative RT-PCR analyses of NSE mRNA in the different tissues of adult geckos, including liver, lung, kidney, heart, spinal cord, brain and ovary of adult geckos. The results revealed that the expression level of NSE mRNA was high in brain, spinal cord and low in heart, while it was not detectable in other tissues; ( C ) real-time qPCR analysis of NSE expression in the spinal cord after transection for the controls (Ctrl) and one, three and seven days post-injury. EF-1α was used for the quantitative normalization. * 1 d vs . Ctrl, p

Techniques Used: Expressing, Northern Blot, Agarose Gel Electrophoresis, Sequencing, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Labeling, Synthesized, In Vitro, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

13) Product Images from "R-Loop Formation In Trans at an AGGAG Repeat"

Article Title: R-Loop Formation In Trans at an AGGAG Repeat

Journal: Journal of Nucleic Acids

doi: 10.1155/2013/629218

R-loop formation in trans detected by radiolabeling of the transcript. (a) Agarose gel stained with ethidium bromide and (b) autoradiogram of the same gel. Lane 1: pBluescript SK(−) linearized with Xba I; lane 2: supercoiled pHC624-(AGGAG) 22 ; lane 3: pHC624-(AGGAG) 22 linearized with Sca I; lane 4: pSK-(AGGAG) 22 linearized with Xba I; lane 5: linearized pSK-(AGGAG) 22 and supercoiled pHC624-(AGGAG) 22 ; and lane 6: linearized pSK-(AGGAG) 22 and linearized pHC624-(AGGAG) 22 . All DNAs were incubated in the transcription mixture with T7 RNA polymerase.
Figure Legend Snippet: R-loop formation in trans detected by radiolabeling of the transcript. (a) Agarose gel stained with ethidium bromide and (b) autoradiogram of the same gel. Lane 1: pBluescript SK(−) linearized with Xba I; lane 2: supercoiled pHC624-(AGGAG) 22 ; lane 3: pHC624-(AGGAG) 22 linearized with Sca I; lane 4: pSK-(AGGAG) 22 linearized with Xba I; lane 5: linearized pSK-(AGGAG) 22 and supercoiled pHC624-(AGGAG) 22 ; and lane 6: linearized pSK-(AGGAG) 22 and linearized pHC624-(AGGAG) 22 . All DNAs were incubated in the transcription mixture with T7 RNA polymerase.

Techniques Used: Radioactivity, Agarose Gel Electrophoresis, Staining, Incubation

R-loop formation on pSK-(AGGAG) 22 and effects of RNase T1 on the formation. A plasmid containing a (AGGAG) 22 repeat was transcribed with T7 RNA polymerase and the effects on the topology of the plasmid were examined by agarose gel electrophoresis. (a) Lane 1: mock transcribed; lane 2: transcribed with T7 RNA polymerase; lane 3: transcribed with T3 RNA polymerase. (b) lane 1: mock transcribed, lane 2: transcribed with T7 RNA polymerase, Lane 3: transcribed with T7 RNA polymerase in the presence of 3 units of RNase T1; lane 4: transcribed with T7 RNA polymerase and ethanol-precipitated; and lane 5: transcribed with T7 RNA polymerase, ethanol-precipitated, and then incubated with 3 units of RNase T1.
Figure Legend Snippet: R-loop formation on pSK-(AGGAG) 22 and effects of RNase T1 on the formation. A plasmid containing a (AGGAG) 22 repeat was transcribed with T7 RNA polymerase and the effects on the topology of the plasmid were examined by agarose gel electrophoresis. (a) Lane 1: mock transcribed; lane 2: transcribed with T7 RNA polymerase; lane 3: transcribed with T3 RNA polymerase. (b) lane 1: mock transcribed, lane 2: transcribed with T7 RNA polymerase, Lane 3: transcribed with T7 RNA polymerase in the presence of 3 units of RNase T1; lane 4: transcribed with T7 RNA polymerase and ethanol-precipitated; and lane 5: transcribed with T7 RNA polymerase, ethanol-precipitated, and then incubated with 3 units of RNase T1.

Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Incubation

R-loop formation in trans and its dependence on supercoiling of the target DNA. RNA containing an AGGAG repeat was produced from linearized pSK-(AGGAG) 22 , and the effects on a T7 promoterless plasmid containing an AGGAG repeat, pHC624-(AGGAG) 22 , were examined by agarose gel electrophoresis. Lane 1: pSK-(AGGAG) 22 linearized with Xba I; lane 2: linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 3: supercoiled pHC624-(AGGAG) 22 , which lacked T7 promoter; lane 4: supercoiled pHC624-(AGGAG) 22 incubated in transcription mixture with T7 RNA polymerase; lane 5: supercoiled pHC624-(AGGAG) 22 and linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 6: supercoiled pHC624-(AGGAG) 22 and pBluescript SK(−) linearized with Xba I, transcribed with T7 RNA polymerase; lane 7: supercoiled pHC624; lane 8: supercoiled pHC624 and linearized pSK-(AGGAG) 22 incubated in transcription mixture with T7 RNA polymerase; lane 9: pHC624-(AGGAG) 22 relaxed with vaccinia topoisomerase I; lane 10: relaxed pHC624-(AGAAG) 22 and linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 11: same as lane 5 and ethanol-precipitated; lane 12: same as lane 11 and treated with 90 units of E. coli RNase H (TaKaRa) in 40 mM Tris · HCl pH 7.7, 4 mM MgCl 2 , 1 mM DTT, 4% glycerol, and 0.003% bovine serum albumin at 37°C for 1 h.
Figure Legend Snippet: R-loop formation in trans and its dependence on supercoiling of the target DNA. RNA containing an AGGAG repeat was produced from linearized pSK-(AGGAG) 22 , and the effects on a T7 promoterless plasmid containing an AGGAG repeat, pHC624-(AGGAG) 22 , were examined by agarose gel electrophoresis. Lane 1: pSK-(AGGAG) 22 linearized with Xba I; lane 2: linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 3: supercoiled pHC624-(AGGAG) 22 , which lacked T7 promoter; lane 4: supercoiled pHC624-(AGGAG) 22 incubated in transcription mixture with T7 RNA polymerase; lane 5: supercoiled pHC624-(AGGAG) 22 and linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 6: supercoiled pHC624-(AGGAG) 22 and pBluescript SK(−) linearized with Xba I, transcribed with T7 RNA polymerase; lane 7: supercoiled pHC624; lane 8: supercoiled pHC624 and linearized pSK-(AGGAG) 22 incubated in transcription mixture with T7 RNA polymerase; lane 9: pHC624-(AGGAG) 22 relaxed with vaccinia topoisomerase I; lane 10: relaxed pHC624-(AGAAG) 22 and linearized pSK-(AGGAG) 22 transcribed with T7 RNA polymerase; lane 11: same as lane 5 and ethanol-precipitated; lane 12: same as lane 11 and treated with 90 units of E. coli RNase H (TaKaRa) in 40 mM Tris · HCl pH 7.7, 4 mM MgCl 2 , 1 mM DTT, 4% glycerol, and 0.003% bovine serum albumin at 37°C for 1 h.

Techniques Used: Produced, Plasmid Preparation, Agarose Gel Electrophoresis, Incubation

Related Articles

Synthesized:

Article Title: Northern blot detection of endogenous small RNAs (~14 nt) in bacterial total RNA extracts
Article Snippet: .. For specific detection of B. subtilis and E. coli 5S rRNA loading controls and for E. coli 6S RNA, antisense transcripts covering the respective full-length RNA and internally labeled with digoxigenin-UTP were synthesized from PCR templates by T7 RNA polymerase (according to the DIG RNA Labeling Mix protocol provided by Roche Diagnostics, Mannheim, Germany). .. For native PAGE, samples were adjusted to 1× native loading buffer by mixing with 6× native loading buffer [0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol blue, 30% (v/v) glycerol].

Labeling:

Article Title: Northern blot detection of endogenous small RNAs (~14 nt) in bacterial total RNA extracts
Article Snippet: .. For specific detection of B. subtilis and E. coli 5S rRNA loading controls and for E. coli 6S RNA, antisense transcripts covering the respective full-length RNA and internally labeled with digoxigenin-UTP were synthesized from PCR templates by T7 RNA polymerase (according to the DIG RNA Labeling Mix protocol provided by Roche Diagnostics, Mannheim, Germany). .. For native PAGE, samples were adjusted to 1× native loading buffer by mixing with 6× native loading buffer [0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol blue, 30% (v/v) glycerol].

Purification:

Article Title: Comprehensive Secondary Structure Elucidation of Four Genera of the Family Pospiviroidae
Article Snippet: .. Preparation of RNA strands In order to produce the viroid transcripts the DNA templates were incubated in the presence of T7 RNA polymerase in a buffer containing 80 mM HEPES-KOH (pH 7.5), 24 mM MgCl2 , 2 mM spermidine, 40 mM DTT, 5 mM of each NTP, 0.004 units/mL pyrophosphatase (Roche Diagnostics), 40 units of RNAseOUT (Life Technologies) and 2 µL of purified T7 RNA polymerase. .. The resulting mixtures were incubated at 37°C for 90 min. Then, DNase RQ1 (2 µL) was added and the sample incubated for 20 min at 37°C.

Article Title: R-Loop Formation In Trans at an AGGAG Repeat
Article Snippet: .. The plasmid, designated pSK-(AGGAG)22 , purified from E. coli and negatively supercoiled, was transcribed with T7 RNA polymerase. .. Agarose gel electrophoresis showed extensive relaxation of the DNA ( , lane 2) indicative of R-loop formation.

Article Title: Human mitochondrial RNA polymerase primes lagging-strand DNA synthesis in vitro
Article Snippet: .. The reaction mixture (25 μl), contained 35 fmol of single-stranded M13mp18 DNA (New England Biolabs), 10 mM Tris·HCl (pH 8.0), 20 mM MgCl2 , 1 mM DTT, 100 μg/ml BSA, 400 μM ATP, 150 μM CTP, and GTP, 10 μM UTP, 0.2 μM α-32 P UTP (3,000 Ci/mmol), 4 units of RNase inhibitor (Amersham Biosciences), and the indicated concentrations of T7 RNA polymerase (Roche) or purified POLRMT protein. ..

Incubation:

Article Title: Comprehensive Secondary Structure Elucidation of Four Genera of the Family Pospiviroidae
Article Snippet: .. Preparation of RNA strands In order to produce the viroid transcripts the DNA templates were incubated in the presence of T7 RNA polymerase in a buffer containing 80 mM HEPES-KOH (pH 7.5), 24 mM MgCl2 , 2 mM spermidine, 40 mM DTT, 5 mM of each NTP, 0.004 units/mL pyrophosphatase (Roche Diagnostics), 40 units of RNAseOUT (Life Technologies) and 2 µL of purified T7 RNA polymerase. .. The resulting mixtures were incubated at 37°C for 90 min. Then, DNase RQ1 (2 µL) was added and the sample incubated for 20 min at 37°C.

other:

Article Title: R-Loop Formation In Trans at an AGGAG Repeat
Article Snippet: Thus, the result indicated that the transcript was separated from and reassociated with the template and that the “separator” function of T7 RNA polymerase was not compromised on the CTCCT-repeat template, in agreement with results reported for other class-switch regions and a telomeric repeat [ ].

Polymerase Chain Reaction:

Article Title: Northern blot detection of endogenous small RNAs (~14 nt) in bacterial total RNA extracts
Article Snippet: .. For specific detection of B. subtilis and E. coli 5S rRNA loading controls and for E. coli 6S RNA, antisense transcripts covering the respective full-length RNA and internally labeled with digoxigenin-UTP were synthesized from PCR templates by T7 RNA polymerase (according to the DIG RNA Labeling Mix protocol provided by Roche Diagnostics, Mannheim, Germany). .. For native PAGE, samples were adjusted to 1× native loading buffer by mixing with 6× native loading buffer [0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol blue, 30% (v/v) glycerol].

Plasmid Preparation:

Article Title: R-Loop Formation In Trans at an AGGAG Repeat
Article Snippet: .. The plasmid, designated pSK-(AGGAG)22 , purified from E. coli and negatively supercoiled, was transcribed with T7 RNA polymerase. .. Agarose gel electrophoresis showed extensive relaxation of the DNA ( , lane 2) indicative of R-loop formation.

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  • 92
    Roche t7 rna polymerase
    Secondary structure presentation of ( A ) mature B. subtilis 6S-1 RNA (190 nt), adapted from ( 11 ), and ( B ) of E. coli 6S RNA according to ( 9 ); both RNAs were transcribed with two artificially added G residues (lowercase letters) for reasons of efficient synthesis by <t>T7</t> RNA polymerase. In both panels, the black lines along the sequence indicate the region of B. subtilis 6S-1 RNA and E. coli 6S RNA that serve as a template for the synthesis of small transcripts (product RNAs = pRNAs) by RNA polymerase (this study, 9); arrows mark the starting point of pRNA transcription; the chemically synthesized pRNA mimics are depicted in the grey boxes on the left above the 6S RNA structures. In panel A, 6S-1 RNA nt 151–157 complementary to nt 2–8 of the LNA probe are boxed. LNA probes for pRNA detection are shown in grey boxes on the right above the secondary structures; DIG, digoxigenin attached to the 5′-end via a C6 linker; small letters depict DNA residues, capital letters LNA residues.
    T7 Rna Polymerase, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 344 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Roche rnase free dnase i
    Analysis of transcript levels in 7 h germlings. A. parasiticus SU-1, AFS10, and Δ veA were grown on YES solid media and spores were collected at 5 days. Germlings were grown from fresh spores in GMS liquid medium for 7 h, frozen in liquid nitrogen, and stored at −80 °C. RNA was extracted from germlings using grinding and sonication as described in Methods. RT-PCR was performed on total RNA treated with <t>RNAse-free</t> <t>DNAse</t> I with primers specific for each gene ( Table 3 ). PCR products were separated on a 1% agarose gel by electrophoresis. Citrate synthase, a constitutively expressed gene, was used as a positive control. gDNA, genomic DNA.
    Rnase Free Dnase I, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 358 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche dnase i
    <t>DNase</t> I footprinting of the yefM-yoeB promoter-operator region. Footprinting reactions were performed as outlined in Materials and Methods using PCR fragments biotinylated at the 5′ ends of either upper or lower strands. YefM concentrations (μM, left to right): 0, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5 and 5.0. YefM–YoeB–His 6 concentrations (μM, left to right): 0, 0.007, 0.018, 0.036, 0.072, 0.18, 0.36, 0.72, 1.8 and 3.6. The locations of the L and S repeats are marked by inverted arrows. Shaded boxes denote the regions protected from DNase I digestion by YefM and YefM–YoeB–His 6 . A + G, Maxam–Gilbert sequencing reactions. A position on the lower strand that is hypersenstive to DNase I cleavage in the presence of YefM and YefM–YoeB–His 6 is highlighted by the star. The relative dispositions of regions on the upper and lower strands that are protected from DNase I digestion and other features of the yefM-yoeB promoter–operator region are illustrated in the lower panel.
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    Secondary structure presentation of ( A ) mature B. subtilis 6S-1 RNA (190 nt), adapted from ( 11 ), and ( B ) of E. coli 6S RNA according to ( 9 ); both RNAs were transcribed with two artificially added G residues (lowercase letters) for reasons of efficient synthesis by T7 RNA polymerase. In both panels, the black lines along the sequence indicate the region of B. subtilis 6S-1 RNA and E. coli 6S RNA that serve as a template for the synthesis of small transcripts (product RNAs = pRNAs) by RNA polymerase (this study, 9); arrows mark the starting point of pRNA transcription; the chemically synthesized pRNA mimics are depicted in the grey boxes on the left above the 6S RNA structures. In panel A, 6S-1 RNA nt 151–157 complementary to nt 2–8 of the LNA probe are boxed. LNA probes for pRNA detection are shown in grey boxes on the right above the secondary structures; DIG, digoxigenin attached to the 5′-end via a C6 linker; small letters depict DNA residues, capital letters LNA residues.

    Journal: Nucleic Acids Research

    Article Title: Northern blot detection of endogenous small RNAs (~14 nt) in bacterial total RNA extracts

    doi: 10.1093/nar/gkq437

    Figure Lengend Snippet: Secondary structure presentation of ( A ) mature B. subtilis 6S-1 RNA (190 nt), adapted from ( 11 ), and ( B ) of E. coli 6S RNA according to ( 9 ); both RNAs were transcribed with two artificially added G residues (lowercase letters) for reasons of efficient synthesis by T7 RNA polymerase. In both panels, the black lines along the sequence indicate the region of B. subtilis 6S-1 RNA and E. coli 6S RNA that serve as a template for the synthesis of small transcripts (product RNAs = pRNAs) by RNA polymerase (this study, 9); arrows mark the starting point of pRNA transcription; the chemically synthesized pRNA mimics are depicted in the grey boxes on the left above the 6S RNA structures. In panel A, 6S-1 RNA nt 151–157 complementary to nt 2–8 of the LNA probe are boxed. LNA probes for pRNA detection are shown in grey boxes on the right above the secondary structures; DIG, digoxigenin attached to the 5′-end via a C6 linker; small letters depict DNA residues, capital letters LNA residues.

    Article Snippet: For specific detection of B. subtilis and E. coli 5S rRNA loading controls and for E. coli 6S RNA, antisense transcripts covering the respective full-length RNA and internally labeled with digoxigenin-UTP were synthesized from PCR templates by T7 RNA polymerase (according to the DIG RNA Labeling Mix protocol provided by Roche Diagnostics, Mannheim, Germany).

    Techniques: Sequencing, Synthesized

    Determination of cis -acting replication elements in the NS5B coding region. (A) Schematic display of T7-based HCV minigenome reporter constructs. HCV minigenome containing the antisense (left) or sense (right) sequence of the Renilla luciferase gene and the antisense sequence of the EMCV IRES flanked by the 5′ end (1 to 377) and differently truncated NS5B coding region-connected 3′ UTR or 3′ UTR alone was juxtaposed precisely at the T7 transcription start site and followed by the HDV ribozyme gene. (B) The indicated reporter vectors were transfected into Huh-NNRZ cells with or without pAM8-1 expressing T7 RNA polymerase. Relative Renilla luciferase activities in the lysates were determined at 48 h posttransfection. The columns and bars represent the means and standard deviations of three independent triplicate transfections. (C) Huh-NNRZ cells were transfected with in vitro transcribed cRLNS5B1 RNA (left) or EMCVRLNS5B1 RNA (right) together with capped firefly luciferase RNA as an internal control. Relative Renilla luciferase activities in the lysates were determined at 24 h posttransfection. Absolute values of Renilla and firefly luciferase activity are listed below the corresponding bars.

    Journal: Journal of Virology

    Article Title: Exploiting cis-Acting Replication Elements To Direct Hepatitis C Virus-Dependent Transgene Expression

    doi: 10.1128/JVI.79.10.5923-5932.2005

    Figure Lengend Snippet: Determination of cis -acting replication elements in the NS5B coding region. (A) Schematic display of T7-based HCV minigenome reporter constructs. HCV minigenome containing the antisense (left) or sense (right) sequence of the Renilla luciferase gene and the antisense sequence of the EMCV IRES flanked by the 5′ end (1 to 377) and differently truncated NS5B coding region-connected 3′ UTR or 3′ UTR alone was juxtaposed precisely at the T7 transcription start site and followed by the HDV ribozyme gene. (B) The indicated reporter vectors were transfected into Huh-NNRZ cells with or without pAM8-1 expressing T7 RNA polymerase. Relative Renilla luciferase activities in the lysates were determined at 48 h posttransfection. The columns and bars represent the means and standard deviations of three independent triplicate transfections. (C) Huh-NNRZ cells were transfected with in vitro transcribed cRLNS5B1 RNA (left) or EMCVRLNS5B1 RNA (right) together with capped firefly luciferase RNA as an internal control. Relative Renilla luciferase activities in the lysates were determined at 24 h posttransfection. Absolute values of Renilla and firefly luciferase activity are listed below the corresponding bars.

    Article Snippet: Plasmids were linearized at SalI site located immediately downstream of the HDV ribozyme, and these fragments were used as templates for runoff RNA synthesis with T7 RNA polymerase according to the protocol supplied by the manufacturer (Roche).

    Techniques: Construct, Sequencing, Luciferase, Transfection, Expressing, In Vitro, Activity Assay

    Analysis of transcript levels in 7 h germlings. A. parasiticus SU-1, AFS10, and Δ veA were grown on YES solid media and spores were collected at 5 days. Germlings were grown from fresh spores in GMS liquid medium for 7 h, frozen in liquid nitrogen, and stored at −80 °C. RNA was extracted from germlings using grinding and sonication as described in Methods. RT-PCR was performed on total RNA treated with RNAse-free DNAse I with primers specific for each gene ( Table 3 ). PCR products were separated on a 1% agarose gel by electrophoresis. Citrate synthase, a constitutively expressed gene, was used as a positive control. gDNA, genomic DNA.

    Journal: Toxins

    Article Title: Aflatoxin Biosynthesis Is a Novel Source of Reactive Oxygen Species—A Potential Redox Signal to Initiate Resistance to Oxidative Stress?

    doi: 10.3390/toxins7051411

    Figure Lengend Snippet: Analysis of transcript levels in 7 h germlings. A. parasiticus SU-1, AFS10, and Δ veA were grown on YES solid media and spores were collected at 5 days. Germlings were grown from fresh spores in GMS liquid medium for 7 h, frozen in liquid nitrogen, and stored at −80 °C. RNA was extracted from germlings using grinding and sonication as described in Methods. RT-PCR was performed on total RNA treated with RNAse-free DNAse I with primers specific for each gene ( Table 3 ). PCR products were separated on a 1% agarose gel by electrophoresis. Citrate synthase, a constitutively expressed gene, was used as a positive control. gDNA, genomic DNA.

    Article Snippet: RT-PCR was performed on total RNA treated with RNAse-free DNAse I (Roche Diagnostics, Indianapolis, IN, USA) with primers specific for the coding region of each gene.

    Techniques: Sonication, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Positive Control

    DNase I footprinting of the yefM-yoeB promoter-operator region. Footprinting reactions were performed as outlined in Materials and Methods using PCR fragments biotinylated at the 5′ ends of either upper or lower strands. YefM concentrations (μM, left to right): 0, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5 and 5.0. YefM–YoeB–His 6 concentrations (μM, left to right): 0, 0.007, 0.018, 0.036, 0.072, 0.18, 0.36, 0.72, 1.8 and 3.6. The locations of the L and S repeats are marked by inverted arrows. Shaded boxes denote the regions protected from DNase I digestion by YefM and YefM–YoeB–His 6 . A + G, Maxam–Gilbert sequencing reactions. A position on the lower strand that is hypersenstive to DNase I cleavage in the presence of YefM and YefM–YoeB–His 6 is highlighted by the star. The relative dispositions of regions on the upper and lower strands that are protected from DNase I digestion and other features of the yefM-yoeB promoter–operator region are illustrated in the lower panel.

    Journal: Nucleic Acids Research

    Article Title: Toxin-antitoxin regulation: bimodal interaction of YefM-YoeB with paired DNA palindromes exerts transcriptional autorepression

    doi: 10.1093/nar/gkl1028

    Figure Lengend Snippet: DNase I footprinting of the yefM-yoeB promoter-operator region. Footprinting reactions were performed as outlined in Materials and Methods using PCR fragments biotinylated at the 5′ ends of either upper or lower strands. YefM concentrations (μM, left to right): 0, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5 and 5.0. YefM–YoeB–His 6 concentrations (μM, left to right): 0, 0.007, 0.018, 0.036, 0.072, 0.18, 0.36, 0.72, 1.8 and 3.6. The locations of the L and S repeats are marked by inverted arrows. Shaded boxes denote the regions protected from DNase I digestion by YefM and YefM–YoeB–His 6 . A + G, Maxam–Gilbert sequencing reactions. A position on the lower strand that is hypersenstive to DNase I cleavage in the presence of YefM and YefM–YoeB–His 6 is highlighted by the star. The relative dispositions of regions on the upper and lower strands that are protected from DNase I digestion and other features of the yefM-yoeB promoter–operator region are illustrated in the lower panel.

    Article Snippet: Each reaction was treated with 0.0075 U of DNase I (Roche, RNase free, 10 U/μl diluted in buffer [20 mM Tris–HCl (pH 7.5), 50 mM NaCl, 7.5 mM MgCl2 and 5 mM CaCl2 ]) for 45 s at 22°C.

    Techniques: Footprinting, Polymerase Chain Reaction, Sequencing