t7 rna polymerase Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs t7 rna polymerase
    Schematic representation of the PATHseq (Preferential Amplification of Pathogenic Sequences) method. ( 1 ) Total mRNAs from clinical sample, including human mRNAs and relatively scarce pathogenic mRNAs; ( 2 ) Total mRNAs are transcribed into first strand cDNAs with P1 primer; ( 3 ) RNase H cleaves RNAs in RNA-DNA duplex; ( 4 ) Reverse transcriptase (RT) synthesizes secondary cDNA strands with P2 primers; ( 5 ) <t>T7</t> RNA polymerase synthesizes RNAs in the presence of T7 promoter; ( 6 ) Synthesized anti-sense RNAs; ( 7 ) Synthesized RNAs are hybridized to human reference (non-pathogenic) cDNA library coated on a solid phase. RNase H cleaves bound RNAs (human RNAs) in RNA-DNA duplex; ( 8 ) Pathogenic RNAs are enriched; ( 9 ) Reverse transcription; ( 10 ) RNase H cleaves RNAs in RNA-DNA duplex; ( 11 ) T7 RNA polymerase synthesizes RNAs; ( 12 ) New RNAs synthesized from enriched pathogenic RNAs are amplified 100-1000 fold.
    T7 Rna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3676 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t7 rna polymerase/product/New England Biolabs
    Average 99 stars, based on 3676 article reviews
    Price from $9.99 to $1999.99
    t7 rna polymerase - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher t7 rna polymerase
    The last 10 nt of Le (nt 34 to 44) are not necessary for correct transcription initiation at the NS1 GS signal. Primer extensions were carried out by using templates of total RNA derived from transfections with minigenome A36 (lane 2) or D36 (lane 3) or oligo(dT)-purified RNA derived from transfections with minigenome A36 (lanes 4 and 5), A57 (lane 6), D36 (lane 7), or D57 (lane 8). Lane 4 is a negative control in which the transfection reaction mixture did not contain L plasmid. Lane 9 is a positive control using miniantigenomic RNA synthesized from C4 plasmid in vitro by <t>T7</t> RNA polymerase. The size of this primer extension product is 1 nt longer than that of A36, consistent with its predicted structure. Lane 1 is a ddC sequencing reaction carried out with minigenome C41 as a template; the position of the first 4 nt (CCCC) of the NS1 GS signal is indicated. Solid arrowheads indicate the positions of cDNAs generated from RNAs initiated at the minigenome 3′ terminus, which are barely detectable in lanes 5 to 8, and a large open arrowhead indicates the position of cDNAs generated from RNAs initiated at the NS1 GS signal.
    T7 Rna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12288 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t7 rna polymerase/product/Thermo Fisher
    Average 99 stars, based on 12288 article reviews
    Price from $9.99 to $1999.99
    t7 rna polymerase - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    99
    Promega t7rna polymerase promoter
    RNase T 1 interferes with R-loop formation on a linearized switch substrate containing four repeats of murine Sγ3. The substrate, pDR3, was linearized with ApaL1 and then transcribed with <t>T7</t> RNA polymerase in the presence of [α- 32 P]UTP.
    T7rna Polymerase Promoter, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t7rna polymerase promoter/product/Promega
    Average 99 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    t7rna polymerase promoter - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    97
    Millipore t7rna polymerase
    Two forms of mMUTYH protein encoded by the alternatively spliced transcripts. ( A ) In vitro translation of type b and type c Mutyh mRNAs. RNAs synthesized from pT7Blue plasmids carrying type b and type c Mutyh cDNA or human MUTYH ) by <t>T7</t> RNA polymerase were translated using rabbit reticulocyte lysate, and translation products were subjected to a western blot analysis with anti-hMUTYH antibody. No template, during in vitro translation, template RNA was omitted. An arrow indicates 50 kDa mMUTYHα encoded by type b mRNA, and an arrowhead indicates the 47 kDa mMUTYHβ encoded by type c mRNA, respectively. ( B ) The detection of two forms of MUTYH in mouse ES cells. Whole cell extracts prepared from wild-type ES cell line, CCE28 cells, MUTYH-null YDK15 cells, and YDKα or YDKβ cells to which an expression construct for type b or type c cDNA was stably introduced, respectively, were subjected to western blot analysis with anti-hMUTYH antibody. An arrow indicates mMUTYHα and an arrowhead indicates mMUTYHβ, respectively. ( C ) The detection of two forms of mMUTYH protein in mouse thymocytes. Thymocyte extracts prepared from two independent wild-type (lanes 1, 2) and MUTYH-null mice ( Mutyh –/– ; lanes 3, 4) were subjected to western blot analysis with anti-hMUTYH antibody (top panel), or with anti-mMUTYHβN (middle panel). An arrow indicates mMUTYHα and an arrowhead indicates mMUTYHβ, respectively. Stained filter with Coomassie Brilliant Blue is shown (bottom) for loading control.
    T7rna Polymerase, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t7rna polymerase/product/Millipore
    Average 97 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    t7rna polymerase - by Bioz Stars, 2020-07
    97/100 stars
      Buy from Supplier

    97
    TaKaRa t7 rna polymerase
    Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, <t>T7</t> RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.
    T7 Rna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 97/100, based on 1049 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t7 rna polymerase/product/TaKaRa
    Average 97 stars, based on 1049 article reviews
    Price from $9.99 to $1999.99
    t7 rna polymerase - by Bioz Stars, 2020-07
    97/100 stars
      Buy from Supplier

    Image Search Results


    Schematic representation of the PATHseq (Preferential Amplification of Pathogenic Sequences) method. ( 1 ) Total mRNAs from clinical sample, including human mRNAs and relatively scarce pathogenic mRNAs; ( 2 ) Total mRNAs are transcribed into first strand cDNAs with P1 primer; ( 3 ) RNase H cleaves RNAs in RNA-DNA duplex; ( 4 ) Reverse transcriptase (RT) synthesizes secondary cDNA strands with P2 primers; ( 5 ) T7 RNA polymerase synthesizes RNAs in the presence of T7 promoter; ( 6 ) Synthesized anti-sense RNAs; ( 7 ) Synthesized RNAs are hybridized to human reference (non-pathogenic) cDNA library coated on a solid phase. RNase H cleaves bound RNAs (human RNAs) in RNA-DNA duplex; ( 8 ) Pathogenic RNAs are enriched; ( 9 ) Reverse transcription; ( 10 ) RNase H cleaves RNAs in RNA-DNA duplex; ( 11 ) T7 RNA polymerase synthesizes RNAs; ( 12 ) New RNAs synthesized from enriched pathogenic RNAs are amplified 100-1000 fold.

    Journal: Scientific Reports

    Article Title: Preferential Amplification of Pathogenic Sequences

    doi: 10.1038/srep11047

    Figure Lengend Snippet: Schematic representation of the PATHseq (Preferential Amplification of Pathogenic Sequences) method. ( 1 ) Total mRNAs from clinical sample, including human mRNAs and relatively scarce pathogenic mRNAs; ( 2 ) Total mRNAs are transcribed into first strand cDNAs with P1 primer; ( 3 ) RNase H cleaves RNAs in RNA-DNA duplex; ( 4 ) Reverse transcriptase (RT) synthesizes secondary cDNA strands with P2 primers; ( 5 ) T7 RNA polymerase synthesizes RNAs in the presence of T7 promoter; ( 6 ) Synthesized anti-sense RNAs; ( 7 ) Synthesized RNAs are hybridized to human reference (non-pathogenic) cDNA library coated on a solid phase. RNase H cleaves bound RNAs (human RNAs) in RNA-DNA duplex; ( 8 ) Pathogenic RNAs are enriched; ( 9 ) Reverse transcription; ( 10 ) RNase H cleaves RNAs in RNA-DNA duplex; ( 11 ) T7 RNA polymerase synthesizes RNAs; ( 12 ) New RNAs synthesized from enriched pathogenic RNAs are amplified 100-1000 fold.

    Article Snippet: Beads were re-suspended in 8.4 μl nuclease-free water, then the following solutions were added: 2 μl of 10x PATH buffer, 2 μl Octamers (10 μM), 1 μl dNTP mix (25 mM), 3.2 μl rNTP mix (25 mM), 0.4 μl Pyrophosphase (Inorganic (E. coli ), 100 units/ml, New England BioLabs, Ipswich, MA), 1 μl reference human cDNAs (0.1 μg/1 μl), 1 μl M-MuLV Reverse Transcriptase (NEB), and 1 μl T7 RNA polymerase (NEB).

    Techniques: Amplification, Synthesized, cDNA Library Assay

    The last 10 nt of Le (nt 34 to 44) are not necessary for correct transcription initiation at the NS1 GS signal. Primer extensions were carried out by using templates of total RNA derived from transfections with minigenome A36 (lane 2) or D36 (lane 3) or oligo(dT)-purified RNA derived from transfections with minigenome A36 (lanes 4 and 5), A57 (lane 6), D36 (lane 7), or D57 (lane 8). Lane 4 is a negative control in which the transfection reaction mixture did not contain L plasmid. Lane 9 is a positive control using miniantigenomic RNA synthesized from C4 plasmid in vitro by T7 RNA polymerase. The size of this primer extension product is 1 nt longer than that of A36, consistent with its predicted structure. Lane 1 is a ddC sequencing reaction carried out with minigenome C41 as a template; the position of the first 4 nt (CCCC) of the NS1 GS signal is indicated. Solid arrowheads indicate the positions of cDNAs generated from RNAs initiated at the minigenome 3′ terminus, which are barely detectable in lanes 5 to 8, and a large open arrowhead indicates the position of cDNAs generated from RNAs initiated at the NS1 GS signal.

    Journal: Journal of Virology

    Article Title: Functional Analysis of the Genomic and Antigenomic Promoters of Human Respiratory Syncytial Virus

    doi:

    Figure Lengend Snippet: The last 10 nt of Le (nt 34 to 44) are not necessary for correct transcription initiation at the NS1 GS signal. Primer extensions were carried out by using templates of total RNA derived from transfections with minigenome A36 (lane 2) or D36 (lane 3) or oligo(dT)-purified RNA derived from transfections with minigenome A36 (lanes 4 and 5), A57 (lane 6), D36 (lane 7), or D57 (lane 8). Lane 4 is a negative control in which the transfection reaction mixture did not contain L plasmid. Lane 9 is a positive control using miniantigenomic RNA synthesized from C4 plasmid in vitro by T7 RNA polymerase. The size of this primer extension product is 1 nt longer than that of A36, consistent with its predicted structure. Lane 1 is a ddC sequencing reaction carried out with minigenome C41 as a template; the position of the first 4 nt (CCCC) of the NS1 GS signal is indicated. Solid arrowheads indicate the positions of cDNAs generated from RNAs initiated at the minigenome 3′ terminus, which are barely detectable in lanes 5 to 8, and a large open arrowhead indicates the position of cDNAs generated from RNAs initiated at the NS1 GS signal.

    Article Snippet: Oligo(dT)-purified or total RNA representing one-third to one-half of a well of cells or 5 pmol of RNA transcribed in vitro from plasmid C4 by T7 RNA polymerase was annealed to an excess of 5′ 32 P-labelled, negative-sense, CAT-specific oligonucleotide probe (5′-GGGATATATCAACGGTGGTATATCCAGTG) in 1× SuperScript II buffer (Life Technologies) by heating the mixture to 95°C for 5 min and placing it at room temperature for 15 min.

    Techniques: Derivative Assay, Transfection, Purification, Negative Control, Plasmid Preparation, Positive Control, Synthesized, In Vitro, Sequencing, Generated

    RNase T 1 interferes with R-loop formation on a linearized switch substrate containing four repeats of murine Sγ3. The substrate, pDR3, was linearized with ApaL1 and then transcribed with T7 RNA polymerase in the presence of [α- 32 P]UTP.

    Journal:

    Article Title: Mechanism of R-Loop Formation at Immunoglobulin Class Switch Sequences ▿Mechanism of R-Loop Formation at Immunoglobulin Class Switch Sequences ▿ †

    doi: 10.1128/MCB.01251-07

    Figure Lengend Snippet: RNase T 1 interferes with R-loop formation on a linearized switch substrate containing four repeats of murine Sγ3. The substrate, pDR3, was linearized with ApaL1 and then transcribed with T7 RNA polymerase in the presence of [α- 32 P]UTP.

    Article Snippet: Moreover, the amount of R-loop formation is similar or higher for all of our buffers containing Li+ , Na+ , K+ , or Cs+ (Fig. , lanes 4 to 7 and lanes 12 to 15 and data not shown) relative to the manufacturer's buffer for T7 RNA polymerase (Fig. , lanes 2 and 10), which is prepared using Na+ (and has the same composition as our Na+ -based transcription buffer).

    Techniques:

    Agarose gel showing self-cleavage of the HHRz in the context of MS2 RNA sequences. Lane 1, DNA marker consisting of 75, 200, 300, 400, 500, 750, 1000, 1500, etc. base-pair fragments. Lane 2 and 3, products of T7 RNA polymerase directed transcription of templates containing the active and inactive (G5A mutant) HHRz, respectively.

    Journal: Virology Journal

    Article Title: A recombinant RNA bacteriophage system to identify functionally important nucleotides in a self-cleaving ribozyme

    doi: 10.1186/1743-422X-11-116

    Figure Lengend Snippet: Agarose gel showing self-cleavage of the HHRz in the context of MS2 RNA sequences. Lane 1, DNA marker consisting of 75, 200, 300, 400, 500, 750, 1000, 1500, etc. base-pair fragments. Lane 2 and 3, products of T7 RNA polymerase directed transcription of templates containing the active and inactive (G5A mutant) HHRz, respectively.

    Article Snippet: After linearization of the resulting plasmids with ApaI and purification of the DNA, RNA was synthesized by T7 RNA polymerase transcription using the RiboMAX Large Scale RNA Production System (Promega).

    Techniques: Agarose Gel Electrophoresis, Marker, Mutagenesis

    Cis-expression of R434 ribozyme inhibits HPV-16E6/E7 in vitro translation. ( A ) Map of HPV-16E6/E7 cis-expression constructs with R434 and R434i ribozymes. PCR-amplified fragments containing the entire HPV-16 E6/E7 genes (nucleotides 97–868) linked to R434 (pCR16E6/E7RZ) or R434i (pCR16E6/E7RZi) ribozymes were cloned in the pCR3.1 vector. The pCR16HH plasmid contains only the HPV-16E6/E7 genes. The relative positions of the Sty I sites used for cloning the ribozymes and the vector poly(A) signal are shown. ( B ) The protein products produced by plasmids pCR16HH, pCR16E6/E7RZ, and pCR16E6/E7RZi were examined by in vitro translation reactions using T7 RNA polymerase and rabbit reticulocyte lysates in the presence of [ 35 S]methionine. ←, the position of E6 and E7 proteins. Luc, luciferase protein reaction control.

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

    Article Title: Inhibition of HPV-16 E6/E7 immortalization of normal keratinocytes by hairpin ribozymes

    doi:

    Figure Lengend Snippet: Cis-expression of R434 ribozyme inhibits HPV-16E6/E7 in vitro translation. ( A ) Map of HPV-16E6/E7 cis-expression constructs with R434 and R434i ribozymes. PCR-amplified fragments containing the entire HPV-16 E6/E7 genes (nucleotides 97–868) linked to R434 (pCR16E6/E7RZ) or R434i (pCR16E6/E7RZi) ribozymes were cloned in the pCR3.1 vector. The pCR16HH plasmid contains only the HPV-16E6/E7 genes. The relative positions of the Sty I sites used for cloning the ribozymes and the vector poly(A) signal are shown. ( B ) The protein products produced by plasmids pCR16HH, pCR16E6/E7RZ, and pCR16E6/E7RZi were examined by in vitro translation reactions using T7 RNA polymerase and rabbit reticulocyte lysates in the presence of [ 35 S]methionine. ←, the position of E6 and E7 proteins. Luc, luciferase protein reaction control.

    Article Snippet: HPV-16 E6/E7 proteins were produced using the T7 RNA polymerase promoter from pCR16HH, pCR16E6/E7RZ, and pCR16E6/E7RZi and using an in vitro transcription–translation system (rabbit reticulocyte TnT, Promega) and [35 S]methionine (Amersham).

    Techniques: Expressing, In Vitro, Construct, Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Produced, Luciferase

    Two forms of mMUTYH protein encoded by the alternatively spliced transcripts. ( A ) In vitro translation of type b and type c Mutyh mRNAs. RNAs synthesized from pT7Blue plasmids carrying type b and type c Mutyh cDNA or human MUTYH ) by T7 RNA polymerase were translated using rabbit reticulocyte lysate, and translation products were subjected to a western blot analysis with anti-hMUTYH antibody. No template, during in vitro translation, template RNA was omitted. An arrow indicates 50 kDa mMUTYHα encoded by type b mRNA, and an arrowhead indicates the 47 kDa mMUTYHβ encoded by type c mRNA, respectively. ( B ) The detection of two forms of MUTYH in mouse ES cells. Whole cell extracts prepared from wild-type ES cell line, CCE28 cells, MUTYH-null YDK15 cells, and YDKα or YDKβ cells to which an expression construct for type b or type c cDNA was stably introduced, respectively, were subjected to western blot analysis with anti-hMUTYH antibody. An arrow indicates mMUTYHα and an arrowhead indicates mMUTYHβ, respectively. ( C ) The detection of two forms of mMUTYH protein in mouse thymocytes. Thymocyte extracts prepared from two independent wild-type (lanes 1, 2) and MUTYH-null mice ( Mutyh –/– ; lanes 3, 4) were subjected to western blot analysis with anti-hMUTYH antibody (top panel), or with anti-mMUTYHβN (middle panel). An arrow indicates mMUTYHα and an arrowhead indicates mMUTYHβ, respectively. Stained filter with Coomassie Brilliant Blue is shown (bottom) for loading control.

    Journal: Nucleic Acids Research

    Article Title: Identification and characterization of two forms of mouse MUTYH proteins encoded by alternatively spliced transcripts

    doi: 10.1093/nar/gkh214

    Figure Lengend Snippet: Two forms of mMUTYH protein encoded by the alternatively spliced transcripts. ( A ) In vitro translation of type b and type c Mutyh mRNAs. RNAs synthesized from pT7Blue plasmids carrying type b and type c Mutyh cDNA or human MUTYH ) by T7 RNA polymerase were translated using rabbit reticulocyte lysate, and translation products were subjected to a western blot analysis with anti-hMUTYH antibody. No template, during in vitro translation, template RNA was omitted. An arrow indicates 50 kDa mMUTYHα encoded by type b mRNA, and an arrowhead indicates the 47 kDa mMUTYHβ encoded by type c mRNA, respectively. ( B ) The detection of two forms of MUTYH in mouse ES cells. Whole cell extracts prepared from wild-type ES cell line, CCE28 cells, MUTYH-null YDK15 cells, and YDKα or YDKβ cells to which an expression construct for type b or type c cDNA was stably introduced, respectively, were subjected to western blot analysis with anti-hMUTYH antibody. An arrow indicates mMUTYHα and an arrowhead indicates mMUTYHβ, respectively. ( C ) The detection of two forms of mMUTYH protein in mouse thymocytes. Thymocyte extracts prepared from two independent wild-type (lanes 1, 2) and MUTYH-null mice ( Mutyh –/– ; lanes 3, 4) were subjected to western blot analysis with anti-hMUTYH antibody (top panel), or with anti-mMUTYHβN (middle panel). An arrow indicates mMUTYHα and an arrowhead indicates mMUTYHβ, respectively. Stained filter with Coomassie Brilliant Blue is shown (bottom) for loading control.

    Article Snippet: The transcripts were synthesized from BamHI linearized plasmids by T7 RNA polymerase at 30°C for 15 min using Single Tube Protein System 2 (Novagen).

    Techniques: In Vitro, Synthesized, Western Blot, Expressing, Construct, Stable Transfection, Mouse Assay, Staining

    Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.

    Article Snippet: Assay of the TTcDR reaction The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara).

    Techniques: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Autoradiography, Marker, Purification

    Schematic representation of the transcription- and translation-coupled DNA replication system. Circular DNA encoding phi29 DNA polymerase under control of the T7 promoter is incubated with the reconstituted translation system including T7 RNA polymerase. mRNA is transcribed from the DNA, and phi29 DNA polymerase is translated. The polymerase attaches to the circular DNA and initiates the polymerization of a long single-stranded RNA in a rolling-circle manner. The polymerase further synthesizes the complementary strand to produce double-stranded DNA, which is a long repeat of the circular DNA sequence. The next round of transcription and translation occurs from the double-stranded DNA.

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Schematic representation of the transcription- and translation-coupled DNA replication system. Circular DNA encoding phi29 DNA polymerase under control of the T7 promoter is incubated with the reconstituted translation system including T7 RNA polymerase. mRNA is transcribed from the DNA, and phi29 DNA polymerase is translated. The polymerase attaches to the circular DNA and initiates the polymerization of a long single-stranded RNA in a rolling-circle manner. The polymerase further synthesizes the complementary strand to produce double-stranded DNA, which is a long repeat of the circular DNA sequence. The next round of transcription and translation occurs from the double-stranded DNA.

    Article Snippet: Assay of the TTcDR reaction The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara).

    Techniques: Incubation, Sequencing