m7g 5 ppp ppp 5 g rna cap structure analog  (New England Biolabs)


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    Name:
    m7G 5 ppp 5 G RNA Cap Structure Analog
    Description:
    m7G 5 ppp 5 G RNA Cap Structure Analog 5 umol
    Catalog Number:
    S1404L
    Price:
    616
    Size:
    5 umol
    Category:
    Capping Reagents for DNA RNA Synthesis
    Score:
    85
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    Structured Review

    New England Biolabs m7g 5 ppp ppp 5 g rna cap structure analog
    m7G 5 ppp 5 G RNA Cap Structure Analog
    m7G 5 ppp 5 G RNA Cap Structure Analog 5 umol
    https://www.bioz.com/result/m7g 5 ppp ppp 5 g rna cap structure analog/product/New England Biolabs
    Average 99 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    m7g 5 ppp ppp 5 g rna cap structure analog - by Bioz Stars, 2019-10
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    Related Articles

    Clone Assay:

    Article Title: Cap analog and Potato virus A HC-Pro silencing suppressor improve GFP transient expression using an infectious virus vector in Nicotiana benthamiana
    Article Snippet: Finally, these clones were checked by restriction analysis with Xba I and Sac I restriction enzymes. .. In vitro transcription with T7 RNA polymerase: Complete in vitro transcription reaction mixtures contained 5 to 10 µg of linearized DNA template plus nuclease free water; 25 mM (each) ATP, CTP, and UTP; 2 mM GTP; 40 mM cap analog m7G(5') ppp (5')G (New England Biolabs); 10µl of T7 RNA polymerase; and 20 µl T7 transcription buffer (Promega) in a total volume of 100 µl.

    Article Title: Effect of Alternating Passage on Adaptation of Sindbis Virus to Vertebrate and Invertebrate Cells
    Article Snippet: The pTR339 clone ( ) is more representative of wild-type SINV and is virulent for young mice. .. Virus stocks were prepared from both clones by linearization with XhoI, and RNA was transcribed in the presence of M7G-5′-ppp-5′-G cap (New England Biolabs, Beverly, MA) with SP6 RNA polymerase (Invitrogen, Carlsbad, CA), followed by electroporation of BHK cells using standard methods. .. Three different types of passage series were done for each SINV strain: (i) serial (specialized) passage in BHK cells; (ii) serial (specialized) passage in C6/36 cells; and (iii) alternating passage between BHK and C6/36 cells.

    Centrifugation:

    Article Title: Human Cytomegalovirus UL40 Signal Peptide Regulates Cell Surface Expression of the Natural Killer Cell Ligands HLA-E and gpUL18
    Article Snippet: The PCR products were transcribed in vitro with SP6 RNA polymerase at 42 °C in the presence of 500 μM m7G(5′)ppp(5′)G CAP analogue (New England Biolabs). mRNAs were translated in 25 μl reticulocyte lysate (Promega) containing [35] S-methionine and [35] S-cysteine, and two equivalents of nuclease-treated rough microsomes prepared from dog pancreas ( )or digitonin-permeabilized 721.221 cells (105 cells) as indicated. .. 10 μM (Z-LL)2 -ketone or 30 μM N-nenzoyl-Asn-Leu-Thr-methylamide was added to inhibit SPP or N-glycosylation respectively( ).

    Luciferase:

    Article Title: Adaptor Protein 1A Facilitates Dengue Virus Replication
    Article Snippet: pRL-SV40 vector (Promega), which contains Renilla luciferase gene, was linearized with Xba I. .. One μg of purified DNA was subjected to in vitro transcription using the RiboMAX Large Scale RNA Production System-T7 (Promega) in the presence of 20 mM m7 G(5′)ppp(5′)G RNA cap structure analog (New England BioLabs, Ipswich, MA, USA) and resultant RNA product was purified using RNeasy Mini Kit (QIAGEN, Hilden, Germany).

    Reporter Assay:

    Article Title: Adaptor Protein 1A Facilitates Dengue Virus Replication
    Article Snippet: One μg of purified DNA was subjected to in vitro transcription using the RiboMAX Large Scale RNA Production System-T7 (Promega) in the presence of 20 mM m7 G(5′)ppp(5′)G RNA cap structure analog (New England BioLabs, Ipswich, MA, USA) and resultant RNA product was purified using RNeasy Mini Kit (QIAGEN, Hilden, Germany). .. After the second round of siRNA transfection, cells were transfected with 2.5 nM reporter RNA using Lipofectamine 2000 (Invitrogen) followed by replacement with fresh culture medium at 4 h later.

    Mass Spectrometry:

    Article Title: Novel Insect-Specific Eilat Virus-Based Chimeric Vaccine Candidates Provide Durable, Mono- and Multivalent, Single-Dose Protection against Lethal Alphavirus Challenge
    Article Snippet: Linearized cDNAs were purified via phenol-chloroform extraction, and ∼1 μg was utilized for each transcription reaction: 0.5 mM ribonucleoside triphosphate (rNTP), 0.5 mM m7G(5′)ppp(5′)G RNA cap (NEB), 0.5 μl RNase inhibitor, and 1.25 μl of SP6 polymerase (Ambion, Carlsbad, CA) in a 25-μl total volume. .. Monolayers were either trypsinized (vertebrate cells) or gently scraped (C7/10 cells) into single-cell suspensions, washed 5 times with phosphate-buffered saline (PBS), and resuspended in 450 μl or 700 μl (Vero cells only) of PBS.

    Synthesized:

    Article Title: A Viral mRNA Motif at the 3′-Untranslated Region that Confers Translatability in a Cell-Specific Manner. Implications for Virus Evolution
    Article Snippet: Reactions containing 12.5 ng/μl of template DNA, 1000 U/ml of RNA polymerase, 1× RNAPol Reaction Buffer, 1 mM m7 G(5′)ppp(5′)G RNA Cap Structure Analog (New England Biolabs), 0.5 mM ATP, 0.5 mM CTP, 0.5 mM UTP, 0.25 mM GTP, and 0.32 U/μl RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen) were incubated for 4 h at 37 °C. .. Reactions containing 12.5 ng/μl of template DNA, 1000 U/ml of RNA polymerase, 1× RNAPol Reaction Buffer, 1 mM m7 G(5′)ppp(5′)G RNA Cap Structure Analog (New England Biolabs), 0.5 mM ATP, 0.5 mM CTP, 0.5 mM UTP, 0.25 mM GTP, and 0.32 U/μl RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen) were incubated for 4 h at 37 °C.

    Article Title: Plant-Produced Cottontail Rabbit Papillomavirus L1 Protein Protects against Tumor Challenge: a Proof-of-Concept Study
    Article Snippet: DNA from transformed N. tabacum plants was screened by PCR for the CRPV L1 gene using the same primer pair and conditions as those used for the initial amplification of the gene. .. Transcripts were synthesized in vitro using the T7 RNA polymerase (RiboMAX large-scale RNA production system T7; Promega) and capped by addition of the RNA cap structure analogue m7G(5)ppp(5)G (New England Biolabs). .. Inoculation of 3-week-old N. benthamiana plants and subsequent monitoring of infection were done as described previously ( ).

    Article Title: Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA
    Article Snippet: RNA was synthesized using a MEGAscript T7 kit (Ambion, Carlsbad, CA, USA) with purified tail PCR product. .. We also used a Cap/NTP mixture with an m7G(5′)ppp(5′)G ARCA cap analog (New England Biolabs, Manchester, CT, USA), 3′-methylcytidine triphosphate and pseudo-uridine triphosphate (TriLink Biotechnologies, San Diego, CA, USA) following the protocol for generation of modified mRNA.

    Article Title: hnRNP A1 Interacts with the 5? Untranslated Regions of Enterovirus 71 and Sindbis Virus RNA and Is Required for Viral Replication
    Article Snippet: The positive-strand RNA was synthesized by in vitro transcription using T7 polymerase and labeled with [α-32 P]GTP. .. The RNA was capped by adding an m7G(5′)ppp(5′)G cap structural analog (NEB) (final concentration, 5 mM) into the reaction mixture and purified as described above.

    Construct:

    Article Title: Cap analog and Potato virus A HC-Pro silencing suppressor improve GFP transient expression using an infectious virus vector in Nicotiana benthamiana
    Article Snippet: In vitro transcription with T7 RNA polymerase: Complete in vitro transcription reaction mixtures contained 5 to 10 µg of linearized DNA template plus nuclease free water; 25 mM (each) ATP, CTP, and UTP; 2 mM GTP; 40 mM cap analog m7G(5') ppp (5')G (New England Biolabs); 10µl of T7 RNA polymerase; and 20 µl T7 transcription buffer (Promega) in a total volume of 100 µl. .. The concentration of RNA was estimated by UV absorption following ethidium bromide staining.

    Article Title: A Simple and Efficient In Vivo Non-viral RNA Transfection Method for Labeling the Whole Axonal Tree of Individual Adult Long-Range Projection Neurons
    Article Snippet: Paragraph title: RNA Constructs ... Finally, DNA template in vitro transcription was carried out using 1.5 μg of plasmidic DNA in a 50 μl distilled DEPC water solution containing 10× SP6 buffer (5 μl), rNTP mix (5 μl) (Amersham Pharmacia, UK), CAP analog (m7G(5′)ppp(5′)G) (5 μl) (New England Biolabs, Ipswich, MA, USA), Rnasin (1.5 μl) (Promega, Madison, WI, USA) and SP6 RNA polymerase (0.5 μl; New England Biolabs, Ipswich, MA, USA).

    Electrophoresis:

    Article Title: Characterization of Synthetic Chikungunya Viruses Based on the Consensus Sequence of Recent E1-226V Isolates
    Article Snippet: RNA was transcribed from plasmids with the phi2.5 promoter using the AmpliScribe T7 high yield transcription kit (Epicenter), the m7 GpppA RNA cap structure analog (NEB) and 0.7 µg of template DNA that had been linearized with Spe I. .. After a 3-h reaction at 37°C, template DNA was digested with DnaseI and RNA was purified by precipitation with 7.5 M LiCl (Ambion).

    Article Title: A Simple and Efficient In Vivo Non-viral RNA Transfection Method for Labeling the Whole Axonal Tree of Individual Adult Long-Range Projection Neurons
    Article Snippet: After checking digestion by electrophoresis in 0.7% agarose gel, DNA templates were purified and precipitated. .. Finally, DNA template in vitro transcription was carried out using 1.5 μg of plasmidic DNA in a 50 μl distilled DEPC water solution containing 10× SP6 buffer (5 μl), rNTP mix (5 μl) (Amersham Pharmacia, UK), CAP analog (m7G(5′)ppp(5′)G) (5 μl) (New England Biolabs, Ipswich, MA, USA), Rnasin (1.5 μl) (Promega, Madison, WI, USA) and SP6 RNA polymerase (0.5 μl; New England Biolabs, Ipswich, MA, USA).

    Incubation:

    Article Title: Characterization of Synthetic Chikungunya Viruses Based on the Consensus Sequence of Recent E1-226V Isolates
    Article Snippet: RNA was transcribed from plasmids with the phi2.5 promoter using the AmpliScribe T7 high yield transcription kit (Epicenter), the m7 GpppA RNA cap structure analog (NEB) and 0.7 µg of template DNA that had been linearized with Spe I. .. RNA was transcribed from plasmids with the phi2.5 promoter using the AmpliScribe T7 high yield transcription kit (Epicenter), the m7 GpppA RNA cap structure analog (NEB) and 0.7 µg of template DNA that had been linearized with Spe I.

    Article Title: Cap analog and Potato virus A HC-Pro silencing suppressor improve GFP transient expression using an infectious virus vector in Nicotiana benthamiana
    Article Snippet: Linearization and purification of pPZP212 containing TCV-sGFP: Plasmid containing full-length TCV-sGFP was linearized by cleavage with Xba ׀ enzyme, and incubated 1 hour at 37°C. .. In vitro transcription with T7 RNA polymerase: Complete in vitro transcription reaction mixtures contained 5 to 10 µg of linearized DNA template plus nuclease free water; 25 mM (each) ATP, CTP, and UTP; 2 mM GTP; 40 mM cap analog m7G(5') ppp (5')G (New England Biolabs); 10µl of T7 RNA polymerase; and 20 µl T7 transcription buffer (Promega) in a total volume of 100 µl.

    Article Title: Effect of Alternating Passage on Adaptation of Sindbis Virus to Vertebrate and Invertebrate Cells
    Article Snippet: Virus stocks were prepared from both clones by linearization with XhoI, and RNA was transcribed in the presence of M7G-5′-ppp-5′-G cap (New England Biolabs, Beverly, MA) with SP6 RNA polymerase (Invitrogen, Carlsbad, CA), followed by electroporation of BHK cells using standard methods. .. Virus stocks were prepared from both clones by linearization with XhoI, and RNA was transcribed in the presence of M7G-5′-ppp-5′-G cap (New England Biolabs, Beverly, MA) with SP6 RNA polymerase (Invitrogen, Carlsbad, CA), followed by electroporation of BHK cells using standard methods.

    Article Title: A Viral mRNA Motif at the 3′-Untranslated Region that Confers Translatability in a Cell-Specific Manner. Implications for Virus Evolution
    Article Snippet: Plasmids were linearized with XhoI (or NotI in the case of all plasmids derived from pnsP-LUC) and used as templates for in vitro RNA transcription with T7 or SP6 RNA polymerase (New England Biolabs). .. Reactions containing 12.5 ng/μl of template DNA, 1000 U/ml of RNA polymerase, 1× RNAPol Reaction Buffer, 1 mM m7 G(5′)ppp(5′)G RNA Cap Structure Analog (New England Biolabs), 0.5 mM ATP, 0.5 mM CTP, 0.5 mM UTP, 0.25 mM GTP, and 0.32 U/μl RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen) were incubated for 4 h at 37 °C. .. The template DNA was then digested with 0.1 U/μl RNase-free Recombinant DNase I (Takara) for 10 minutes at 37 °C.

    Article Title: Structural mutants of dengue virus 2 transmembrane domains exhibit host-range phenotype
    Article Snippet: Transcripts were generated for each DV2 mutant clone using the RiboMAX™ Large Scale RNA Product Systems for T7 RNA Polymerase (Promega) following manufacturer's instructions, with the addition of RNA cap analog 7 mg(ppp)G (NEB # S1404S). .. Transcripts were generated for each DV2 mutant clone using the RiboMAX™ Large Scale RNA Product Systems for T7 RNA Polymerase (Promega) following manufacturer's instructions, with the addition of RNA cap analog 7 mg(ppp)G (NEB # S1404S).

    Article Title: Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA
    Article Snippet: We also used a Cap/NTP mixture with an m7G(5′)ppp(5′)G ARCA cap analog (New England Biolabs, Manchester, CT, USA), 3′-methylcytidine triphosphate and pseudo-uridine triphosphate (TriLink Biotechnologies, San Diego, CA, USA) following the protocol for generation of modified mRNA. .. We also used a Cap/NTP mixture with an m7G(5′)ppp(5′)G ARCA cap analog (New England Biolabs, Manchester, CT, USA), 3′-methylcytidine triphosphate and pseudo-uridine triphosphate (TriLink Biotechnologies, San Diego, CA, USA) following the protocol for generation of modified mRNA.

    Article Title: hnRNP A1 Interacts with the 5? Untranslated Regions of Enterovirus 71 and Sindbis Virus RNA and Is Required for Viral Replication
    Article Snippet: The RNA was capped by adding an m7G(5′)ppp(5′)G cap structural analog (NEB) (final concentration, 5 mM) into the reaction mixture and purified as described above. .. The RNA was capped by adding an m7G(5′)ppp(5′)G cap structural analog (NEB) (final concentration, 5 mM) into the reaction mixture and purified as described above.

    Amplification:

    Article Title: Human Cytomegalovirus UL40 Signal Peptide Regulates Cell Surface Expression of the Natural Killer Cell Ligands HLA-E and gpUL18
    Article Snippet: To generate templates for in vitro transcription, UL40 with a hybrid HLA-C/UL40 signal peptide (MNKFSNTRIGFTCAVMAPRTLVLLLSGALALTQTWA, to generate pAL303) and HLA-A2 with a hybrid HCMV AD169 UL40/HLA-A2 signal peptide (MNKFSNTRIGFTCAVMAPRTLVLLLSGALALTQTWA, to generate pAL307) were amplified by PCR using suitable plasmid templates. .. The PCR products were transcribed in vitro with SP6 RNA polymerase at 42 °C in the presence of 500 μM m7G(5′)ppp(5′)G CAP analogue (New England Biolabs). mRNAs were translated in 25 μl reticulocyte lysate (Promega) containing [35] S-methionine and [35] S-cysteine, and two equivalents of nuclease-treated rough microsomes prepared from dog pancreas ( )or digitonin-permeabilized 721.221 cells (105 cells) as indicated.

    Article Title: A Simple and Efficient In Vivo Non-viral RNA Transfection Method for Labeling the Whole Axonal Tree of Individual Adult Long-Range Projection Neurons
    Article Snippet: A cDNA template of the construct (pSinRep5-Pal-eGFP) was first amplified using competent E. coli XL1Blue bacteria. .. Finally, DNA template in vitro transcription was carried out using 1.5 μg of plasmidic DNA in a 50 μl distilled DEPC water solution containing 10× SP6 buffer (5 μl), rNTP mix (5 μl) (Amersham Pharmacia, UK), CAP analog (m7G(5′)ppp(5′)G) (5 μl) (New England Biolabs, Ipswich, MA, USA), Rnasin (1.5 μl) (Promega, Madison, WI, USA) and SP6 RNA polymerase (0.5 μl; New England Biolabs, Ipswich, MA, USA).

    Article Title: hnRNP A1 Interacts with the 5? Untranslated Regions of Enterovirus 71 and Sindbis Virus RNA and Is Required for Viral Replication
    Article Snippet: The 3′-terminal 45 nt of SV negative-strand RNA was amplified by PCR from pToto, the infectious clone of SV, using a positive-sense primer containing the T7 promoter and the first 20 nt of SV sequence and a negative-sense primer containing SP6 promoter and nt 33 to 45 of SV. .. The RNA was capped by adding an m7G(5′)ppp(5′)G cap structural analog (NEB) (final concentration, 5 mM) into the reaction mixture and purified as described above.

    Cell Culture:

    Article Title: Effect of Alternating Passage on Adaptation of Sindbis Virus to Vertebrate and Invertebrate Cells
    Article Snippet: Virus stocks were prepared from both clones by linearization with XhoI, and RNA was transcribed in the presence of M7G-5′-ppp-5′-G cap (New England Biolabs, Beverly, MA) with SP6 RNA polymerase (Invitrogen, Carlsbad, CA), followed by electroporation of BHK cells using standard methods. .. Infected cell cultures were incubated at 32°C to eliminate temperature sensitivity as a factor in the outcomes.

    Expressing:

    Article Title: Adaptor Protein 1A Facilitates Dengue Virus Replication
    Article Snippet: One μg of purified DNA was subjected to in vitro transcription using the RiboMAX Large Scale RNA Production System-T7 (Promega) in the presence of 20 mM m7 G(5′)ppp(5′)G RNA cap structure analog (New England BioLabs, Ipswich, MA, USA) and resultant RNA product was purified using RNeasy Mini Kit (QIAGEN, Hilden, Germany). .. After the second round of siRNA transfection, cells were transfected with 2.5 nM reporter RNA using Lipofectamine 2000 (Invitrogen) followed by replacement with fresh culture medium at 4 h later.

    Article Title: A Simple and Efficient In Vivo Non-viral RNA Transfection Method for Labeling the Whole Axonal Tree of Individual Adult Long-Range Projection Neurons
    Article Snippet: An RNA construct engineered to drive the expression of an enhanced variety of the green fluorescent protein from Aqueoria victoria (eGFP) fusioned with a palmytoilation motif from the growth associated protein 43 (GAP43) under the Sindbis viral subgenomic promoter ( ) was used in this study. .. Finally, DNA template in vitro transcription was carried out using 1.5 μg of plasmidic DNA in a 50 μl distilled DEPC water solution containing 10× SP6 buffer (5 μl), rNTP mix (5 μl) (Amersham Pharmacia, UK), CAP analog (m7G(5′)ppp(5′)G) (5 μl) (New England Biolabs, Ipswich, MA, USA), Rnasin (1.5 μl) (Promega, Madison, WI, USA) and SP6 RNA polymerase (0.5 μl; New England Biolabs, Ipswich, MA, USA).

    Article Title: Novel Insect-Specific Eilat Virus-Based Chimeric Vaccine Candidates Provide Durable, Mono- and Multivalent, Single-Dose Protection against Lethal Alphavirus Challenge
    Article Snippet: Linearized cDNAs were purified via phenol-chloroform extraction, and ∼1 μg was utilized for each transcription reaction: 0.5 mM ribonucleoside triphosphate (rNTP), 0.5 mM m7G(5′)ppp(5′)G RNA cap (NEB), 0.5 μl RNase inhibitor, and 1.25 μl of SP6 polymerase (Ambion, Carlsbad, CA) in a 25-μl total volume. .. Electroporation conditions (Vero cells only) were 250 V, pulse length of 10 ms, interval between pulses of 1 s, and number of pulses of 3.

    Modification:

    Article Title: Hybrid alphavirus–rhabdovirus propagating replicon particles are versatile and potent vaccine vectors
    Article Snippet: To generate the infectious RNA genome of the propagating replicons, pSFV1-G and pSFVG-gp140 plasmids were linearized with SpeI and transcribed for 2 h at 37°C in a 40-μl reaction mixture. .. The reaction was a modification of the Ampliscribe SP6 transcription kit (Epicentre technologies) containing SP6 reaction buffer, 5 mM each of ATP, CTP, and UTP, 1 mM GTP, 4 mM m7 G(ppp)G RNA cap analog (NEB S1404L), 10 mM DTT, and 2 μl of SP6 polymerase. .. The transcription reactions were stored at −80°C.

    Article Title: Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA
    Article Snippet: RNA was synthesized using a MEGAscript T7 kit (Ambion, Carlsbad, CA, USA) with purified tail PCR product. .. We also used a Cap/NTP mixture with an m7G(5′)ppp(5′)G ARCA cap analog (New England Biolabs, Manchester, CT, USA), 3′-methylcytidine triphosphate and pseudo-uridine triphosphate (TriLink Biotechnologies, San Diego, CA, USA) following the protocol for generation of modified mRNA. .. To generate unmodified mRNA, we made a Cap/NTP mixture using ATP, CTP, UTP, and GTP components.

    Derivative Assay:

    Article Title: A Viral mRNA Motif at the 3′-Untranslated Region that Confers Translatability in a Cell-Specific Manner. Implications for Virus Evolution
    Article Snippet: Plasmids were linearized with XhoI (or NotI in the case of all plasmids derived from pnsP-LUC) and used as templates for in vitro RNA transcription with T7 or SP6 RNA polymerase (New England Biolabs). .. Reactions containing 12.5 ng/μl of template DNA, 1000 U/ml of RNA polymerase, 1× RNAPol Reaction Buffer, 1 mM m7 G(5′)ppp(5′)G RNA Cap Structure Analog (New England Biolabs), 0.5 mM ATP, 0.5 mM CTP, 0.5 mM UTP, 0.25 mM GTP, and 0.32 U/μl RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen) were incubated for 4 h at 37 °C.

    Electroporation:

    Article Title: Effect of Alternating Passage on Adaptation of Sindbis Virus to Vertebrate and Invertebrate Cells
    Article Snippet: The pTR339 clone ( ) is more representative of wild-type SINV and is virulent for young mice. .. Virus stocks were prepared from both clones by linearization with XhoI, and RNA was transcribed in the presence of M7G-5′-ppp-5′-G cap (New England Biolabs, Beverly, MA) with SP6 RNA polymerase (Invitrogen, Carlsbad, CA), followed by electroporation of BHK cells using standard methods. .. Three different types of passage series were done for each SINV strain: (i) serial (specialized) passage in BHK cells; (ii) serial (specialized) passage in C6/36 cells; and (iii) alternating passage between BHK and C6/36 cells.

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy
    Article Snippet: The in vitro transcription reaction was carried out with 10 μg of linearized plasmid DNA in a total volume of 100 μL containing 20 μL 5× RRL buffer (400 mM HEPES (pH 7.5), 60 mM MgCl2 , 10 mM spermidine and 200 mM DTT), NTP-Mix (3.125 mM ATP, CTP and UTP and 1.56 mM GTP), 1 U/μL RNasin (Promega, Madison, WI, USA), 2 U/μL T7 RNA polymerase (New England Biolabs) and 1 mM anti-reverse cap analogue (ARCA; 3′-O-Me- m7G(5′)ppp(5′)G; New England Biolabs). .. The integrity and size of the RNAs was validated by agarose gel electrophoresis.

    Article Title: Novel Insect-Specific Eilat Virus-Based Chimeric Vaccine Candidates Provide Durable, Mono- and Multivalent, Single-Dose Protection against Lethal Alphavirus Challenge
    Article Snippet: Paragraph title: Transcription and electroporation conditions for virus rescue. ... Linearized cDNAs were purified via phenol-chloroform extraction, and ∼1 μg was utilized for each transcription reaction: 0.5 mM ribonucleoside triphosphate (rNTP), 0.5 mM m7G(5′)ppp(5′)G RNA cap (NEB), 0.5 μl RNase inhibitor, and 1.25 μl of SP6 polymerase (Ambion, Carlsbad, CA) in a 25-μl total volume.

    Transfection:

    Article Title: Characterization of Synthetic Chikungunya Viruses Based on the Consensus Sequence of Recent E1-226V Isolates
    Article Snippet: Paragraph title: In vitro Transcription and RNA Transfection ... RNA was transcribed from plasmids with the phi2.5 promoter using the AmpliScribe T7 high yield transcription kit (Epicenter), the m7 GpppA RNA cap structure analog (NEB) and 0.7 µg of template DNA that had been linearized with Spe I.

    Article Title: Adaptor Protein 1A Facilitates Dengue Virus Replication
    Article Snippet: One μg of purified DNA was subjected to in vitro transcription using the RiboMAX Large Scale RNA Production System-T7 (Promega) in the presence of 20 mM m7 G(5′)ppp(5′)G RNA cap structure analog (New England BioLabs, Ipswich, MA, USA) and resultant RNA product was purified using RNeasy Mini Kit (QIAGEN, Hilden, Germany). .. To determine the effect of AP-1A knockdown on translation, Huh7 cells were transfected twice with AP-1A-specific siRNA or control siRNA in a 96-well plate within a 24-h interval using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

    Article Title: The Initiation Factors eIF2, eIF2A, eIF2D, eIF4A, and eIF4G Are Not Involved in Translation Driven by Hepatitis C Virus IRES in Human Cells
    Article Snippet: Linearized plasmids were used as templates for in vitro RNA transcription using T7 or T3 RNA polymerases (New England Biolabs, Ipswich, MA, United States), the m7G(5′)ppp(5′)G cap analog (New England Biolabs) was used for Cap.βGlobin-Luc transcription. .. In vitro -synthesized RNAs were treated with recombinant DNase I (RNase-free) (Takara Bio USA Inc., Terra Bella, CA, United States) for 30 min at 37°C.

    Article Title: A Viral mRNA Motif at the 3′-Untranslated Region that Confers Translatability in a Cell-Specific Manner. Implications for Virus Evolution
    Article Snippet: Paragraph title: In vitro transcription and transfection ... Reactions containing 12.5 ng/μl of template DNA, 1000 U/ml of RNA polymerase, 1× RNAPol Reaction Buffer, 1 mM m7 G(5′)ppp(5′)G RNA Cap Structure Analog (New England Biolabs), 0.5 mM ATP, 0.5 mM CTP, 0.5 mM UTP, 0.25 mM GTP, and 0.32 U/μl RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen) were incubated for 4 h at 37 °C.

    Article Title: Hybrid alphavirus–rhabdovirus propagating replicon particles are versatile and potent vaccine vectors
    Article Snippet: Paragraph title: Transcription of RNA and Transfection to Generate Infectious Particles. ... The reaction was a modification of the Ampliscribe SP6 transcription kit (Epicentre technologies) containing SP6 reaction buffer, 5 mM each of ATP, CTP, and UTP, 1 mM GTP, 4 mM m7 G(ppp)G RNA cap analog (NEB S1404L), 10 mM DTT, and 2 μl of SP6 polymerase.

    Article Title: Structural mutants of dengue virus 2 transmembrane domains exhibit host-range phenotype
    Article Snippet: Paragraph title: In vitro transcription and RNA transfection ... Transcripts were generated for each DV2 mutant clone using the RiboMAX™ Large Scale RNA Product Systems for T7 RNA Polymerase (Promega) following manufacturer's instructions, with the addition of RNA cap analog 7 mg(ppp)G (NEB # S1404S).

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy
    Article Snippet: Paragraph title: 2.5. In Vitro Transcription and RNA Transfection ... The in vitro transcription reaction was carried out with 10 μg of linearized plasmid DNA in a total volume of 100 μL containing 20 μL 5× RRL buffer (400 mM HEPES (pH 7.5), 60 mM MgCl2 , 10 mM spermidine and 200 mM DTT), NTP-Mix (3.125 mM ATP, CTP and UTP and 1.56 mM GTP), 1 U/μL RNasin (Promega, Madison, WI, USA), 2 U/μL T7 RNA polymerase (New England Biolabs) and 1 mM anti-reverse cap analogue (ARCA; 3′-O-Me- m7G(5′)ppp(5′)G; New England Biolabs).

    Article Title: RNAe: an effective method for targeted protein translation enhancement by artificial non-coding RNA with SINEB2 repeat
    Article Snippet: Paragraph title: Cell transfection ... RNA with 5' cap was transcribed also by HiScribe™ T7 High Yield RNA Synthesis Kit with m7G(5′)ppp(5′)G (New England Biolabs, UK) added.

    Concentration Assay:

    Article Title: Characterization of Synthetic Chikungunya Viruses Based on the Consensus Sequence of Recent E1-226V Isolates
    Article Snippet: RNA was transcribed from plasmids with the phi2.5 promoter using the AmpliScribe T7 high yield transcription kit (Epicenter), the m7 GpppA RNA cap structure analog (NEB) and 0.7 µg of template DNA that had been linearized with Spe I. .. After a 3-h reaction at 37°C, template DNA was digested with DnaseI and RNA was purified by precipitation with 7.5 M LiCl (Ambion).

    Article Title: Cap analog and Potato virus A HC-Pro silencing suppressor improve GFP transient expression using an infectious virus vector in Nicotiana benthamiana
    Article Snippet: In vitro transcription with T7 RNA polymerase: Complete in vitro transcription reaction mixtures contained 5 to 10 µg of linearized DNA template plus nuclease free water; 25 mM (each) ATP, CTP, and UTP; 2 mM GTP; 40 mM cap analog m7G(5') ppp (5')G (New England Biolabs); 10µl of T7 RNA polymerase; and 20 µl T7 transcription buffer (Promega) in a total volume of 100 µl. .. The reaction mixtures were incubated at 37°C for 2h, and RNA product was analyzed by agarose gel electrophoresis followed by ethidium bromide staining.

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy
    Article Snippet: The in vitro transcription reaction was carried out with 10 μg of linearized plasmid DNA in a total volume of 100 μL containing 20 μL 5× RRL buffer (400 mM HEPES (pH 7.5), 60 mM MgCl2 , 10 mM spermidine and 200 mM DTT), NTP-Mix (3.125 mM ATP, CTP and UTP and 1.56 mM GTP), 1 U/μL RNasin (Promega, Madison, WI, USA), 2 U/μL T7 RNA polymerase (New England Biolabs) and 1 mM anti-reverse cap analogue (ARCA; 3′-O-Me- m7G(5′)ppp(5′)G; New England Biolabs). .. The integrity and size of the RNAs was validated by agarose gel electrophoresis.

    Article Title: Genome Editing in Mouse Spermatogonial Stem/Progenitor Cells Using Engineered Nucleases
    Article Snippet: First, GFP-ZFN2 and GFP-ZFN1, encoded on the SP202A and SP202B plasmids, were linearized with Xba I restriction enzyme; GFP-TALEN1 and GFP-TALEN2, encoded on M733L and M733R plasmids, were linearized with Afl II. .. T7 MEGAscript kit (Life Technologies, Grand Island, NY) was used with a reduced GTP concentration (1.5 mM) and 6 mM m7 G(5')ppp(5')G RNA Cap Structure Analog (New England Biolabs, Ipswich, MA) added to synthesize RNA. .. Then the Poly(A) Tailing and MEGAClear kits (Life Technologies, Grand Island, NY) were used according to the manufacturer's directions.

    Article Title: hnRNP A1 Interacts with the 5? Untranslated Regions of Enterovirus 71 and Sindbis Virus RNA and Is Required for Viral Replication
    Article Snippet: The positive-strand RNA was synthesized by in vitro transcription using T7 polymerase and labeled with [α-32 P]GTP. .. The RNA was capped by adding an m7G(5′)ppp(5′)G cap structural analog (NEB) (final concentration, 5 mM) into the reaction mixture and purified as described above. .. An EMSA was carried out to determine the interaction between the viral RNA and hnRNP A1 as described previously ( ).

    Infection:

    Article Title: Characterization of Synthetic Chikungunya Viruses Based on the Consensus Sequence of Recent E1-226V Isolates
    Article Snippet: RNA was transcribed from plasmids with the phi2.5 promoter using the AmpliScribe T7 high yield transcription kit (Epicenter), the m7 GpppA RNA cap structure analog (NEB) and 0.7 µg of template DNA that had been linearized with Spe I. .. RNA was transcribed from plasmids with the phi2.5 promoter using the AmpliScribe T7 high yield transcription kit (Epicenter), the m7 GpppA RNA cap structure analog (NEB) and 0.7 µg of template DNA that had been linearized with Spe I.

    Article Title: Effect of Alternating Passage on Adaptation of Sindbis Virus to Vertebrate and Invertebrate Cells
    Article Snippet: Virus stocks were prepared from both clones by linearization with XhoI, and RNA was transcribed in the presence of M7G-5′-ppp-5′-G cap (New England Biolabs, Beverly, MA) with SP6 RNA polymerase (Invitrogen, Carlsbad, CA), followed by electroporation of BHK cells using standard methods. .. Virus stocks were prepared from both clones by linearization with XhoI, and RNA was transcribed in the presence of M7G-5′-ppp-5′-G cap (New England Biolabs, Beverly, MA) with SP6 RNA polymerase (Invitrogen, Carlsbad, CA), followed by electroporation of BHK cells using standard methods.

    Generated:

    Article Title: Dual Function for U2AF35 in AG-Dependent Pre-mRNA Splicing
    Article Snippet: Transcription templates were generated by PCR using plasmids harboring the sequences of AdML, IgM M1-M2 (pμM), or mutant derivatives of both substrates (Fig. ) preceded by an SP6 promoter. .. Full-length substrates were transcribed in the presence of a CAP analog [m7G (5′) ppp (5′) G] (New England Biolabs) and [α-32 P]UTP (Amersham) as described previously ( ).

    Article Title: Structural mutants of dengue virus 2 transmembrane domains exhibit host-range phenotype
    Article Snippet: All DV2 deletion mutant clones were confirmed by sequence analysis (Eurofins MWG Operon, Huntsville, AL). .. Transcripts were generated for each DV2 mutant clone using the RiboMAX™ Large Scale RNA Product Systems for T7 RNA Polymerase (Promega) following manufacturer's instructions, with the addition of RNA cap analog 7 mg(ppp)G (NEB # S1404S). .. The RNA transcripts were transfected into Vero and C6/36 cells as follows: Cells were pelleted and washed in RNase free electroporation buffer (PBS-D for Vero and MOPS for C6/36) and resuspended in their respective buffers at a concentration of 1 × 107 to 5 × 107 cells/ml.

    Article Title: hnRNP A1 Interacts with the 5? Untranslated Regions of Enterovirus 71 and Sindbis Virus RNA and Is Required for Viral Replication
    Article Snippet: RNA probes for use in RNA gel mobility shift assays (EMSAs) were generated by runoff transcription using bacteriophage T7 RNA polymerase and then purified by use of an RNeasy minikit (Qiagen) and labeled at their 5′ ends by using T4 polynucleotide kinase and [γ-32 P]ATP. .. The RNA was capped by adding an m7G(5′)ppp(5′)G cap structural analog (NEB) (final concentration, 5 mM) into the reaction mixture and purified as described above.

    Polymerase Chain Reaction:

    Article Title: Dual Function for U2AF35 in AG-Dependent Pre-mRNA Splicing
    Article Snippet: Transcription templates were generated by PCR using plasmids harboring the sequences of AdML, IgM M1-M2 (pμM), or mutant derivatives of both substrates (Fig. ) preceded by an SP6 promoter. .. Full-length substrates were transcribed in the presence of a CAP analog [m7G (5′) ppp (5′) G] (New England Biolabs) and [α-32 P]UTP (Amersham) as described previously ( ).

    Article Title: Human Cytomegalovirus UL40 Signal Peptide Regulates Cell Surface Expression of the Natural Killer Cell Ligands HLA-E and gpUL18
    Article Snippet: The forward primer introduced the SP6 promoter and a Kozak initiation sequence, while the reverse primer inserted a stop codon at the desired position of the open reading frame. .. The PCR products were transcribed in vitro with SP6 RNA polymerase at 42 °C in the presence of 500 μM m7G(5′)ppp(5′)G CAP analogue (New England Biolabs). mRNAs were translated in 25 μl reticulocyte lysate (Promega) containing [35] S-methionine and [35] S-cysteine, and two equivalents of nuclease-treated rough microsomes prepared from dog pancreas ( )or digitonin-permeabilized 721.221 cells (105 cells) as indicated. .. 10 μM (Z-LL)2 -ketone or 30 μM N-nenzoyl-Asn-Leu-Thr-methylamide was added to inhibit SPP or N-glycosylation respectively( ).

    Article Title: Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA
    Article Snippet: RNA was synthesized using a MEGAscript T7 kit (Ambion, Carlsbad, CA, USA) with purified tail PCR product. .. We also used a Cap/NTP mixture with an m7G(5′)ppp(5′)G ARCA cap analog (New England Biolabs, Manchester, CT, USA), 3′-methylcytidine triphosphate and pseudo-uridine triphosphate (TriLink Biotechnologies, San Diego, CA, USA) following the protocol for generation of modified mRNA.

    Article Title: hnRNP A1 Interacts with the 5? Untranslated Regions of Enterovirus 71 and Sindbis Virus RNA and Is Required for Viral Replication
    Article Snippet: The 3′-terminal 45 nt of SV negative-strand RNA was amplified by PCR from pToto, the infectious clone of SV, using a positive-sense primer containing the T7 promoter and the first 20 nt of SV sequence and a negative-sense primer containing SP6 promoter and nt 33 to 45 of SV. .. The RNA was capped by adding an m7G(5′)ppp(5′)G cap structural analog (NEB) (final concentration, 5 mM) into the reaction mixture and purified as described above.

    Sonication:

    Article Title: Hybrid alphavirus–rhabdovirus propagating replicon particles are versatile and potent vaccine vectors
    Article Snippet: The reaction was a modification of the Ampliscribe SP6 transcription kit (Epicentre technologies) containing SP6 reaction buffer, 5 mM each of ATP, CTP, and UTP, 1 mM GTP, 4 mM m7 G(ppp)G RNA cap analog (NEB S1404L), 10 mM DTT, and 2 μl of SP6 polymerase. .. They were then transfected with 60 μl of transcription reaction in 9 ml of serum-free DMEM containing 90 μl of a cationic liposome reagent containing dimethy–dioctadecyl ammonium bromide ( ) as described ( ).

    Recombinant:

    Article Title: A Viral mRNA Motif at the 3′-Untranslated Region that Confers Translatability in a Cell-Specific Manner. Implications for Virus Evolution
    Article Snippet: Plasmids were linearized with XhoI (or NotI in the case of all plasmids derived from pnsP-LUC) and used as templates for in vitro RNA transcription with T7 or SP6 RNA polymerase (New England Biolabs). .. Reactions containing 12.5 ng/μl of template DNA, 1000 U/ml of RNA polymerase, 1× RNAPol Reaction Buffer, 1 mM m7 G(5′)ppp(5′)G RNA Cap Structure Analog (New England Biolabs), 0.5 mM ATP, 0.5 mM CTP, 0.5 mM UTP, 0.25 mM GTP, and 0.32 U/μl RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen) were incubated for 4 h at 37 °C. .. The template DNA was then digested with 0.1 U/μl RNase-free Recombinant DNase I (Takara) for 10 minutes at 37 °C.

    Fluorescence:

    Article Title: Novel Insect-Specific Eilat Virus-Based Chimeric Vaccine Candidates Provide Durable, Mono- and Multivalent, Single-Dose Protection against Lethal Alphavirus Challenge
    Article Snippet: Linearized cDNAs were purified via phenol-chloroform extraction, and ∼1 μg was utilized for each transcription reaction: 0.5 mM ribonucleoside triphosphate (rNTP), 0.5 mM m7G(5′)ppp(5′)G RNA cap (NEB), 0.5 μl RNase inhibitor, and 1.25 μl of SP6 polymerase (Ambion, Carlsbad, CA) in a 25-μl total volume. .. Electroporation conditions (Vero cells only) were 250 V, pulse length of 10 ms, interval between pulses of 1 s, and number of pulses of 3.

    Mutagenesis:

    Article Title: Dual Function for U2AF35 in AG-Dependent Pre-mRNA Splicing
    Article Snippet: Transcription templates were generated by PCR using plasmids harboring the sequences of AdML, IgM M1-M2 (pμM), or mutant derivatives of both substrates (Fig. ) preceded by an SP6 promoter. .. Full-length substrates were transcribed in the presence of a CAP analog [m7G (5′) ppp (5′) G] (New England Biolabs) and [α-32 P]UTP (Amersham) as described previously ( ).

    Article Title: Structural mutants of dengue virus 2 transmembrane domains exhibit host-range phenotype
    Article Snippet: All DV2 deletion mutant clones were confirmed by sequence analysis (Eurofins MWG Operon, Huntsville, AL). .. Transcripts were generated for each DV2 mutant clone using the RiboMAX™ Large Scale RNA Product Systems for T7 RNA Polymerase (Promega) following manufacturer's instructions, with the addition of RNA cap analog 7 mg(ppp)G (NEB # S1404S). .. The RNA transcripts were transfected into Vero and C6/36 cells as follows: Cells were pelleted and washed in RNase free electroporation buffer (PBS-D for Vero and MOPS for C6/36) and resuspended in their respective buffers at a concentration of 1 × 107 to 5 × 107 cells/ml.

    Isolation:

    Article Title: Human Cytomegalovirus UL40 Signal Peptide Regulates Cell Surface Expression of the Natural Killer Cell Ligands HLA-E and gpUL18
    Article Snippet: The PCR products were transcribed in vitro with SP6 RNA polymerase at 42 °C in the presence of 500 μM m7G(5′)ppp(5′)G CAP analogue (New England Biolabs). mRNAs were translated in 25 μl reticulocyte lysate (Promega) containing [35] S-methionine and [35] S-cysteine, and two equivalents of nuclease-treated rough microsomes prepared from dog pancreas ( )or digitonin-permeabilized 721.221 cells (105 cells) as indicated. .. 10 μM (Z-LL)2 -ketone or 30 μM N-nenzoyl-Asn-Leu-Thr-methylamide was added to inhibit SPP or N-glycosylation respectively( ).

    Microscopy:

    Article Title: Novel Insect-Specific Eilat Virus-Based Chimeric Vaccine Candidates Provide Durable, Mono- and Multivalent, Single-Dose Protection against Lethal Alphavirus Challenge
    Article Snippet: Linearized cDNAs were purified via phenol-chloroform extraction, and ∼1 μg was utilized for each transcription reaction: 0.5 mM ribonucleoside triphosphate (rNTP), 0.5 mM m7G(5′)ppp(5′)G RNA cap (NEB), 0.5 μl RNase inhibitor, and 1.25 μl of SP6 polymerase (Ambion, Carlsbad, CA) in a 25-μl total volume. .. Electroporation conditions (Vero cells only) were 250 V, pulse length of 10 ms, interval between pulses of 1 s, and number of pulses of 3.

    Purification:

    Article Title: Adaptor Protein 1A Facilitates Dengue Virus Replication
    Article Snippet: pRL-SV40 vector (Promega), which contains Renilla luciferase gene, was linearized with Xba I. .. One μg of purified DNA was subjected to in vitro transcription using the RiboMAX Large Scale RNA Production System-T7 (Promega) in the presence of 20 mM m7 G(5′)ppp(5′)G RNA cap structure analog (New England BioLabs, Ipswich, MA, USA) and resultant RNA product was purified using RNeasy Mini Kit (QIAGEN, Hilden, Germany). .. To determine the effect of AP-1A knockdown on translation, Huh7 cells were transfected twice with AP-1A-specific siRNA or control siRNA in a 96-well plate within a 24-h interval using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

    Article Title: Cap analog and Potato virus A HC-Pro silencing suppressor improve GFP transient expression using an infectious virus vector in Nicotiana benthamiana
    Article Snippet: Linearization and purification of pPZP212 containing TCV-sGFP: Plasmid containing full-length TCV-sGFP was linearized by cleavage with Xba ׀ enzyme, and incubated 1 hour at 37°C. .. In vitro transcription with T7 RNA polymerase: Complete in vitro transcription reaction mixtures contained 5 to 10 µg of linearized DNA template plus nuclease free water; 25 mM (each) ATP, CTP, and UTP; 2 mM GTP; 40 mM cap analog m7G(5') ppp (5')G (New England Biolabs); 10µl of T7 RNA polymerase; and 20 µl T7 transcription buffer (Promega) in a total volume of 100 µl.

    Article Title: A Viral mRNA Motif at the 3′-Untranslated Region that Confers Translatability in a Cell-Specific Manner. Implications for Virus Evolution
    Article Snippet: Reactions containing 12.5 ng/μl of template DNA, 1000 U/ml of RNA polymerase, 1× RNAPol Reaction Buffer, 1 mM m7 G(5′)ppp(5′)G RNA Cap Structure Analog (New England Biolabs), 0.5 mM ATP, 0.5 mM CTP, 0.5 mM UTP, 0.25 mM GTP, and 0.32 U/μl RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen) were incubated for 4 h at 37 °C. .. The template DNA was then digested with 0.1 U/μl RNase-free Recombinant DNase I (Takara) for 10 minutes at 37 °C.

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy
    Article Snippet: Briefly, synZIKV sequences were linearized with XhoI (located at the end of the 3′UTR of the viral genome) and the DNA purified with the Nucleo-Spin Extract II kit (Macherey-Nagel, Düren, Germany). .. The in vitro transcription reaction was carried out with 10 μg of linearized plasmid DNA in a total volume of 100 μL containing 20 μL 5× RRL buffer (400 mM HEPES (pH 7.5), 60 mM MgCl2 , 10 mM spermidine and 200 mM DTT), NTP-Mix (3.125 mM ATP, CTP and UTP and 1.56 mM GTP), 1 U/μL RNasin (Promega, Madison, WI, USA), 2 U/μL T7 RNA polymerase (New England Biolabs) and 1 mM anti-reverse cap analogue (ARCA; 3′-O-Me- m7G(5′)ppp(5′)G; New England Biolabs).

    Article Title: Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA
    Article Snippet: RNA was synthesized using a MEGAscript T7 kit (Ambion, Carlsbad, CA, USA) with purified tail PCR product. .. We also used a Cap/NTP mixture with an m7G(5′)ppp(5′)G ARCA cap analog (New England Biolabs, Manchester, CT, USA), 3′-methylcytidine triphosphate and pseudo-uridine triphosphate (TriLink Biotechnologies, San Diego, CA, USA) following the protocol for generation of modified mRNA.

    Article Title: Novel Insect-Specific Eilat Virus-Based Chimeric Vaccine Candidates Provide Durable, Mono- and Multivalent, Single-Dose Protection against Lethal Alphavirus Challenge
    Article Snippet: Ten micrograms of EILV chimera cDNAs was linearized using a NotI restriction site engineered immediately after the poly(A) tail. .. Linearized cDNAs were purified via phenol-chloroform extraction, and ∼1 μg was utilized for each transcription reaction: 0.5 mM ribonucleoside triphosphate (rNTP), 0.5 mM m7G(5′)ppp(5′)G RNA cap (NEB), 0.5 μl RNase inhibitor, and 1.25 μl of SP6 polymerase (Ambion, Carlsbad, CA) in a 25-μl total volume. .. Monolayers were either trypsinized (vertebrate cells) or gently scraped (C7/10 cells) into single-cell suspensions, washed 5 times with phosphate-buffered saline (PBS), and resuspended in 450 μl or 700 μl (Vero cells only) of PBS.

    Article Title: hnRNP A1 Interacts with the 5? Untranslated Regions of Enterovirus 71 and Sindbis Virus RNA and Is Required for Viral Replication
    Article Snippet: The positive-strand RNA was synthesized by in vitro transcription using T7 polymerase and labeled with [α-32 P]GTP. .. The RNA was capped by adding an m7G(5′)ppp(5′)G cap structural analog (NEB) (final concentration, 5 mM) into the reaction mixture and purified as described above. .. An EMSA was carried out to determine the interaction between the viral RNA and hnRNP A1 as described previously ( ).

    Sequencing:

    Article Title: Dual Function for U2AF35 in AG-Dependent Pre-mRNA Splicing
    Article Snippet: The same reverse primers were used for the 3′-half substrates in combination with a forward primer containing the T7 promoter followed by a sequence annealing approximately 20 nucleotides upstream of the BP. .. Full-length substrates were transcribed in the presence of a CAP analog [m7G (5′) ppp (5′) G] (New England Biolabs) and [α-32 P]UTP (Amersham) as described previously ( ).

    Article Title: Human Cytomegalovirus UL40 Signal Peptide Regulates Cell Surface Expression of the Natural Killer Cell Ligands HLA-E and gpUL18
    Article Snippet: The forward primer introduced the SP6 promoter and a Kozak initiation sequence, while the reverse primer inserted a stop codon at the desired position of the open reading frame. .. The PCR products were transcribed in vitro with SP6 RNA polymerase at 42 °C in the presence of 500 μM m7G(5′)ppp(5′)G CAP analogue (New England Biolabs). mRNAs were translated in 25 μl reticulocyte lysate (Promega) containing [35] S-methionine and [35] S-cysteine, and two equivalents of nuclease-treated rough microsomes prepared from dog pancreas ( )or digitonin-permeabilized 721.221 cells (105 cells) as indicated.

    Article Title: hnRNP A1 Interacts with the 5? Untranslated Regions of Enterovirus 71 and Sindbis Virus RNA and Is Required for Viral Replication
    Article Snippet: The 3′-terminal 45 nt of SV negative-strand RNA was amplified by PCR from pToto, the infectious clone of SV, using a positive-sense primer containing the T7 promoter and the first 20 nt of SV sequence and a negative-sense primer containing SP6 promoter and nt 33 to 45 of SV. .. The RNA was capped by adding an m7G(5′)ppp(5′)G cap structural analog (NEB) (final concentration, 5 mM) into the reaction mixture and purified as described above.

    Labeling:

    Article Title: hnRNP A1 Interacts with the 5? Untranslated Regions of Enterovirus 71 and Sindbis Virus RNA and Is Required for Viral Replication
    Article Snippet: Paragraph title: Preparation of labeled RNA probes and binding assay. ... The RNA was capped by adding an m7G(5′)ppp(5′)G cap structural analog (NEB) (final concentration, 5 mM) into the reaction mixture and purified as described above.

    Cotransfection:

    Article Title: RNAe: an effective method for targeted protein translation enhancement by artificial non-coding RNA with SINEB2 repeat
    Article Snippet: Unless stated explicitly, the plasmid concentrations used for transfection were as follows: For pRNAe and plasmid expressing target gene co-transfection, 0.2 μg expression plasmid and 1.2 μg of pRNAe plasmid or control plasmid were used per well of 12-well plates; for the expression of the light-chain and heavy-chain of anti-HIV antibody 10E8, 0.2 μg of each and 1.2 μg of pRNAe plasmid or control plasmid were used per well of 12-well plates. .. RNA with 5' cap was transcribed also by HiScribe™ T7 High Yield RNA Synthesis Kit with m7G(5′)ppp(5′)G (New England Biolabs, UK) added.

    Staining:

    Article Title: Cap analog and Potato virus A HC-Pro silencing suppressor improve GFP transient expression using an infectious virus vector in Nicotiana benthamiana
    Article Snippet: In vitro transcription with T7 RNA polymerase: Complete in vitro transcription reaction mixtures contained 5 to 10 µg of linearized DNA template plus nuclease free water; 25 mM (each) ATP, CTP, and UTP; 2 mM GTP; 40 mM cap analog m7G(5') ppp (5')G (New England Biolabs); 10µl of T7 RNA polymerase; and 20 µl T7 transcription buffer (Promega) in a total volume of 100 µl. .. In vitro transcription with T7 RNA polymerase: Complete in vitro transcription reaction mixtures contained 5 to 10 µg of linearized DNA template plus nuclease free water; 25 mM (each) ATP, CTP, and UTP; 2 mM GTP; 40 mM cap analog m7G(5') ppp (5')G (New England Biolabs); 10µl of T7 RNA polymerase; and 20 µl T7 transcription buffer (Promega) in a total volume of 100 µl.

    Article Title: eIF3 targets cell proliferation mRNAs for translational activation or repression
    Article Snippet: After hypotonic-induced swelling for 45 min on ice, cells were homogenized using a syringe attached to a 26G needle until ∼95% of cells burst, as monitored by trypan blue staining. .. 1 mM m7 G(5′)ppp(5′)G RNA cap structure analog (NEB) was added to reactions when indicated.

    Activated Clotting Time Assay:

    Article Title: Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA
    Article Snippet: The forward and reverse primers 5′-TTG GAC CCT CGT ACA GAA GCT AAT ACG-3′ and 5′-T (120)-CTT CCT ACT CAG GCT TTA TTC AAA GAC CA-3′ were used. .. We also used a Cap/NTP mixture with an m7G(5′)ppp(5′)G ARCA cap analog (New England Biolabs, Manchester, CT, USA), 3′-methylcytidine triphosphate and pseudo-uridine triphosphate (TriLink Biotechnologies, San Diego, CA, USA) following the protocol for generation of modified mRNA.

    Mouse Assay:

    Article Title: Effect of Alternating Passage on Adaptation of Sindbis Virus to Vertebrate and Invertebrate Cells
    Article Snippet: The pTR339 clone ( ) is more representative of wild-type SINV and is virulent for young mice. .. Virus stocks were prepared from both clones by linearization with XhoI, and RNA was transcribed in the presence of M7G-5′-ppp-5′-G cap (New England Biolabs, Beverly, MA) with SP6 RNA polymerase (Invitrogen, Carlsbad, CA), followed by electroporation of BHK cells using standard methods.

    SDS Page:

    Article Title: Human Cytomegalovirus UL40 Signal Peptide Regulates Cell Surface Expression of the Natural Killer Cell Ligands HLA-E and gpUL18
    Article Snippet: The PCR products were transcribed in vitro with SP6 RNA polymerase at 42 °C in the presence of 500 μM m7G(5′)ppp(5′)G CAP analogue (New England Biolabs). mRNAs were translated in 25 μl reticulocyte lysate (Promega) containing [35] S-methionine and [35] S-cysteine, and two equivalents of nuclease-treated rough microsomes prepared from dog pancreas ( )or digitonin-permeabilized 721.221 cells (105 cells) as indicated. .. The PCR products were transcribed in vitro with SP6 RNA polymerase at 42 °C in the presence of 500 μM m7G(5′)ppp(5′)G CAP analogue (New England Biolabs). mRNAs were translated in 25 μl reticulocyte lysate (Promega) containing [35] S-methionine and [35] S-cysteine, and two equivalents of nuclease-treated rough microsomes prepared from dog pancreas ( )or digitonin-permeabilized 721.221 cells (105 cells) as indicated.

    Plasmid Preparation:

    Article Title: Adaptor Protein 1A Facilitates Dengue Virus Replication
    Article Snippet: pRL-SV40 vector (Promega), which contains Renilla luciferase gene, was linearized with Xba I. .. One μg of purified DNA was subjected to in vitro transcription using the RiboMAX Large Scale RNA Production System-T7 (Promega) in the presence of 20 mM m7 G(5′)ppp(5′)G RNA cap structure analog (New England BioLabs, Ipswich, MA, USA) and resultant RNA product was purified using RNeasy Mini Kit (QIAGEN, Hilden, Germany).

    Article Title: Cap analog and Potato virus A HC-Pro silencing suppressor improve GFP transient expression using an infectious virus vector in Nicotiana benthamiana
    Article Snippet: Linearized plasmid DNA was extracted with phenol/chloroform and precipitated in the presence of sodium acetate, and dissolved in RNase-free water. .. In vitro transcription with T7 RNA polymerase: Complete in vitro transcription reaction mixtures contained 5 to 10 µg of linearized DNA template plus nuclease free water; 25 mM (each) ATP, CTP, and UTP; 2 mM GTP; 40 mM cap analog m7G(5') ppp (5')G (New England Biolabs); 10µl of T7 RNA polymerase; and 20 µl T7 transcription buffer (Promega) in a total volume of 100 µl.

    Article Title: Human Cytomegalovirus UL40 Signal Peptide Regulates Cell Surface Expression of the Natural Killer Cell Ligands HLA-E and gpUL18
    Article Snippet: To generate templates for in vitro transcription, UL40 with a hybrid HLA-C/UL40 signal peptide (MNKFSNTRIGFTCAVMAPRTLVLLLSGALALTQTWA, to generate pAL303) and HLA-A2 with a hybrid HCMV AD169 UL40/HLA-A2 signal peptide (MNKFSNTRIGFTCAVMAPRTLVLLLSGALALTQTWA, to generate pAL307) were amplified by PCR using suitable plasmid templates. .. The PCR products were transcribed in vitro with SP6 RNA polymerase at 42 °C in the presence of 500 μM m7G(5′)ppp(5′)G CAP analogue (New England Biolabs). mRNAs were translated in 25 μl reticulocyte lysate (Promega) containing [35] S-methionine and [35] S-cysteine, and two equivalents of nuclease-treated rough microsomes prepared from dog pancreas ( )or digitonin-permeabilized 721.221 cells (105 cells) as indicated.

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy
    Article Snippet: Briefly, synZIKV sequences were linearized with XhoI (located at the end of the 3′UTR of the viral genome) and the DNA purified with the Nucleo-Spin Extract II kit (Macherey-Nagel, Düren, Germany). .. The in vitro transcription reaction was carried out with 10 μg of linearized plasmid DNA in a total volume of 100 μL containing 20 μL 5× RRL buffer (400 mM HEPES (pH 7.5), 60 mM MgCl2 , 10 mM spermidine and 200 mM DTT), NTP-Mix (3.125 mM ATP, CTP and UTP and 1.56 mM GTP), 1 U/μL RNasin (Promega, Madison, WI, USA), 2 U/μL T7 RNA polymerase (New England Biolabs) and 1 mM anti-reverse cap analogue (ARCA; 3′-O-Me- m7G(5′)ppp(5′)G; New England Biolabs). .. After incubation at 37 °C for 2.5 h, 1 U/μL T7 RNA polymerase was added followed by additional 2.5 h incubation at 37 °C.

    Article Title: A Simple and Efficient In Vivo Non-viral RNA Transfection Method for Labeling the Whole Axonal Tree of Individual Adult Long-Range Projection Neurons
    Article Snippet: Finally, DNA template in vitro transcription was carried out using 1.5 μg of plasmidic DNA in a 50 μl distilled DEPC water solution containing 10× SP6 buffer (5 μl), rNTP mix (5 μl) (Amersham Pharmacia, UK), CAP analog (m7G(5′)ppp(5′)G) (5 μl) (New England Biolabs, Ipswich, MA, USA), Rnasin (1.5 μl) (Promega, Madison, WI, USA) and SP6 RNA polymerase (0.5 μl; New England Biolabs, Ipswich, MA, USA). .. Finally, DNA template in vitro transcription was carried out using 1.5 μg of plasmidic DNA in a 50 μl distilled DEPC water solution containing 10× SP6 buffer (5 μl), rNTP mix (5 μl) (Amersham Pharmacia, UK), CAP analog (m7G(5′)ppp(5′)G) (5 μl) (New England Biolabs, Ipswich, MA, USA), Rnasin (1.5 μl) (Promega, Madison, WI, USA) and SP6 RNA polymerase (0.5 μl; New England Biolabs, Ipswich, MA, USA).

    Article Title: RNAe: an effective method for targeted protein translation enhancement by artificial non-coding RNA with SINEB2 repeat
    Article Snippet: For pRNAe-Plus experiments, the amount of pRNAe-Plus plasmid was as listed in the figure, while the total DNA amount per well was kept constant by supplementing to 1.5 μg with an appropriate amount of pRNAe-Mock plasmid. .. RNA with 5' cap was transcribed also by HiScribe™ T7 High Yield RNA Synthesis Kit with m7G(5′)ppp(5′)G (New England Biolabs, UK) added.

    Article Title: Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA
    Article Snippet: In brief, plasmid DNAs were used as the template for poly-(A) tail polymerase chain reaction (PCR). .. We also used a Cap/NTP mixture with an m7G(5′)ppp(5′)G ARCA cap analog (New England Biolabs, Manchester, CT, USA), 3′-methylcytidine triphosphate and pseudo-uridine triphosphate (TriLink Biotechnologies, San Diego, CA, USA) following the protocol for generation of modified mRNA.

    Functional Assay:

    Article Title: A Simple and Efficient In Vivo Non-viral RNA Transfection Method for Labeling the Whole Axonal Tree of Individual Adult Long-Range Projection Neurons
    Article Snippet: Since the functional vector is a single-strand RNA, it was necessary to make in vitro transcription from the plasmidic DNA ( Figure ). .. Finally, DNA template in vitro transcription was carried out using 1.5 μg of plasmidic DNA in a 50 μl distilled DEPC water solution containing 10× SP6 buffer (5 μl), rNTP mix (5 μl) (Amersham Pharmacia, UK), CAP analog (m7G(5′)ppp(5′)G) (5 μl) (New England Biolabs, Ipswich, MA, USA), Rnasin (1.5 μl) (Promega, Madison, WI, USA) and SP6 RNA polymerase (0.5 μl; New England Biolabs, Ipswich, MA, USA).

    Binding Assay:

    Article Title: hnRNP A1 Interacts with the 5? Untranslated Regions of Enterovirus 71 and Sindbis Virus RNA and Is Required for Viral Replication
    Article Snippet: Paragraph title: Preparation of labeled RNA probes and binding assay. ... The RNA was capped by adding an m7G(5′)ppp(5′)G cap structural analog (NEB) (final concentration, 5 mM) into the reaction mixture and purified as described above.

    Agarose Gel Electrophoresis:

    Article Title: Characterization of Synthetic Chikungunya Viruses Based on the Consensus Sequence of Recent E1-226V Isolates
    Article Snippet: RNA was transcribed from plasmids with the phi2.5 promoter using the AmpliScribe T7 high yield transcription kit (Epicenter), the m7 GpppA RNA cap structure analog (NEB) and 0.7 µg of template DNA that had been linearized with Spe I. .. After a 3-h reaction at 37°C, template DNA was digested with DnaseI and RNA was purified by precipitation with 7.5 M LiCl (Ambion).

    Article Title: Cap analog and Potato virus A HC-Pro silencing suppressor improve GFP transient expression using an infectious virus vector in Nicotiana benthamiana
    Article Snippet: In vitro transcription with T7 RNA polymerase: Complete in vitro transcription reaction mixtures contained 5 to 10 µg of linearized DNA template plus nuclease free water; 25 mM (each) ATP, CTP, and UTP; 2 mM GTP; 40 mM cap analog m7G(5') ppp (5')G (New England Biolabs); 10µl of T7 RNA polymerase; and 20 µl T7 transcription buffer (Promega) in a total volume of 100 µl. .. In vitro transcription with T7 RNA polymerase: Complete in vitro transcription reaction mixtures contained 5 to 10 µg of linearized DNA template plus nuclease free water; 25 mM (each) ATP, CTP, and UTP; 2 mM GTP; 40 mM cap analog m7G(5') ppp (5')G (New England Biolabs); 10µl of T7 RNA polymerase; and 20 µl T7 transcription buffer (Promega) in a total volume of 100 µl.

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy
    Article Snippet: The in vitro transcription reaction was carried out with 10 μg of linearized plasmid DNA in a total volume of 100 μL containing 20 μL 5× RRL buffer (400 mM HEPES (pH 7.5), 60 mM MgCl2 , 10 mM spermidine and 200 mM DTT), NTP-Mix (3.125 mM ATP, CTP and UTP and 1.56 mM GTP), 1 U/μL RNasin (Promega, Madison, WI, USA), 2 U/μL T7 RNA polymerase (New England Biolabs) and 1 mM anti-reverse cap analogue (ARCA; 3′-O-Me- m7G(5′)ppp(5′)G; New England Biolabs). .. DNA was digested with DNaseI for one hour and RNA was purified by acidic phenol-chloroform extraction and isopropanol precipitation.

    Article Title: A Simple and Efficient In Vivo Non-viral RNA Transfection Method for Labeling the Whole Axonal Tree of Individual Adult Long-Range Projection Neurons
    Article Snippet: After checking digestion by electrophoresis in 0.7% agarose gel, DNA templates were purified and precipitated. .. Finally, DNA template in vitro transcription was carried out using 1.5 μg of plasmidic DNA in a 50 μl distilled DEPC water solution containing 10× SP6 buffer (5 μl), rNTP mix (5 μl) (Amersham Pharmacia, UK), CAP analog (m7G(5′)ppp(5′)G) (5 μl) (New England Biolabs, Ipswich, MA, USA), Rnasin (1.5 μl) (Promega, Madison, WI, USA) and SP6 RNA polymerase (0.5 μl; New England Biolabs, Ipswich, MA, USA).

    In Vitro:

    Article Title: Characterization of Synthetic Chikungunya Viruses Based on the Consensus Sequence of Recent E1-226V Isolates
    Article Snippet: Paragraph title: In vitro Transcription and RNA Transfection ... RNA was transcribed from plasmids with the phi2.5 promoter using the AmpliScribe T7 high yield transcription kit (Epicenter), the m7 GpppA RNA cap structure analog (NEB) and 0.7 µg of template DNA that had been linearized with Spe I.

    Article Title: Adaptor Protein 1A Facilitates Dengue Virus Replication
    Article Snippet: pRL-SV40 vector (Promega), which contains Renilla luciferase gene, was linearized with Xba I. .. One μg of purified DNA was subjected to in vitro transcription using the RiboMAX Large Scale RNA Production System-T7 (Promega) in the presence of 20 mM m7 G(5′)ppp(5′)G RNA cap structure analog (New England BioLabs, Ipswich, MA, USA) and resultant RNA product was purified using RNeasy Mini Kit (QIAGEN, Hilden, Germany). .. To determine the effect of AP-1A knockdown on translation, Huh7 cells were transfected twice with AP-1A-specific siRNA or control siRNA in a 96-well plate within a 24-h interval using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

    Article Title: Cap analog and Potato virus A HC-Pro silencing suppressor improve GFP transient expression using an infectious virus vector in Nicotiana benthamiana
    Article Snippet: Linearized plasmid DNA was extracted with phenol/chloroform and precipitated in the presence of sodium acetate, and dissolved in RNase-free water. .. In vitro transcription with T7 RNA polymerase: Complete in vitro transcription reaction mixtures contained 5 to 10 µg of linearized DNA template plus nuclease free water; 25 mM (each) ATP, CTP, and UTP; 2 mM GTP; 40 mM cap analog m7G(5') ppp (5')G (New England Biolabs); 10µl of T7 RNA polymerase; and 20 µl T7 transcription buffer (Promega) in a total volume of 100 µl. .. In vitro transcription by T7 RNA polymerase (Promega) was carried out in the presence or absence of the cap analog under the reaction condition.

    Article Title: Dual Function for U2AF35 in AG-Dependent Pre-mRNA Splicing
    Article Snippet: Paragraph title: In vitro transcription of splicing substrates. ... Full-length substrates were transcribed in the presence of a CAP analog [m7G (5′) ppp (5′) G] (New England Biolabs) and [α-32 P]UTP (Amersham) as described previously ( ).

    Article Title: Human Cytomegalovirus UL40 Signal Peptide Regulates Cell Surface Expression of the Natural Killer Cell Ligands HLA-E and gpUL18
    Article Snippet: The forward primer introduced the SP6 promoter and a Kozak initiation sequence, while the reverse primer inserted a stop codon at the desired position of the open reading frame. .. The PCR products were transcribed in vitro with SP6 RNA polymerase at 42 °C in the presence of 500 μM m7G(5′)ppp(5′)G CAP analogue (New England Biolabs). mRNAs were translated in 25 μl reticulocyte lysate (Promega) containing [35] S-methionine and [35] S-cysteine, and two equivalents of nuclease-treated rough microsomes prepared from dog pancreas ( )or digitonin-permeabilized 721.221 cells (105 cells) as indicated. .. 10 μM (Z-LL)2 -ketone or 30 μM N-nenzoyl-Asn-Leu-Thr-methylamide was added to inhibit SPP or N-glycosylation respectively( ).

    Article Title: The Initiation Factors eIF2, eIF2A, eIF2D, eIF4A, and eIF4G Are Not Involved in Translation Driven by Hepatitis C Virus IRES in Human Cells
    Article Snippet: All restriction enzymes were purchased from New England Biolabs (Ipswich, MA, United States). .. Linearized plasmids were used as templates for in vitro RNA transcription using T7 or T3 RNA polymerases (New England Biolabs, Ipswich, MA, United States), the m7G(5′)ppp(5′)G cap analog (New England Biolabs) was used for Cap.βGlobin-Luc transcription. .. In vitro -synthesized RNAs were treated with recombinant DNase I (RNase-free) (Takara Bio USA Inc., Terra Bella, CA, United States) for 30 min at 37°C.

    Article Title: A Viral mRNA Motif at the 3′-Untranslated Region that Confers Translatability in a Cell-Specific Manner. Implications for Virus Evolution
    Article Snippet: Paragraph title: In vitro transcription and transfection ... Reactions containing 12.5 ng/μl of template DNA, 1000 U/ml of RNA polymerase, 1× RNAPol Reaction Buffer, 1 mM m7 G(5′)ppp(5′)G RNA Cap Structure Analog (New England Biolabs), 0.5 mM ATP, 0.5 mM CTP, 0.5 mM UTP, 0.25 mM GTP, and 0.32 U/μl RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen) were incubated for 4 h at 37 °C.

    Article Title: Structural mutants of dengue virus 2 transmembrane domains exhibit host-range phenotype
    Article Snippet: Paragraph title: In vitro transcription and RNA transfection ... Transcripts were generated for each DV2 mutant clone using the RiboMAX™ Large Scale RNA Product Systems for T7 RNA Polymerase (Promega) following manufacturer's instructions, with the addition of RNA cap analog 7 mg(ppp)G (NEB # S1404S).

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy
    Article Snippet: Briefly, synZIKV sequences were linearized with XhoI (located at the end of the 3′UTR of the viral genome) and the DNA purified with the Nucleo-Spin Extract II kit (Macherey-Nagel, Düren, Germany). .. The in vitro transcription reaction was carried out with 10 μg of linearized plasmid DNA in a total volume of 100 μL containing 20 μL 5× RRL buffer (400 mM HEPES (pH 7.5), 60 mM MgCl2 , 10 mM spermidine and 200 mM DTT), NTP-Mix (3.125 mM ATP, CTP and UTP and 1.56 mM GTP), 1 U/μL RNasin (Promega, Madison, WI, USA), 2 U/μL T7 RNA polymerase (New England Biolabs) and 1 mM anti-reverse cap analogue (ARCA; 3′-O-Me- m7G(5′)ppp(5′)G; New England Biolabs). .. After incubation at 37 °C for 2.5 h, 1 U/μL T7 RNA polymerase was added followed by additional 2.5 h incubation at 37 °C.

    Article Title: Plant-Produced Cottontail Rabbit Papillomavirus L1 Protein Protects against Tumor Challenge: a Proof-of-Concept Study
    Article Snippet: DNA from transformed N. tabacum plants was screened by PCR for the CRPV L1 gene using the same primer pair and conditions as those used for the initial amplification of the gene. .. Transcripts were synthesized in vitro using the T7 RNA polymerase (RiboMAX large-scale RNA production system T7; Promega) and capped by addition of the RNA cap structure analogue m7G(5)ppp(5)G (New England Biolabs). .. Inoculation of 3-week-old N. benthamiana plants and subsequent monitoring of infection were done as described previously ( ).

    Article Title: Genome Editing in Mouse Spermatogonial Stem/Progenitor Cells Using Engineered Nucleases
    Article Snippet: For mRNA synthesis in vitro transcription, capping and tailing were performed similar to Warren et al. . .. T7 MEGAscript kit (Life Technologies, Grand Island, NY) was used with a reduced GTP concentration (1.5 mM) and 6 mM m7 G(5')ppp(5')G RNA Cap Structure Analog (New England Biolabs, Ipswich, MA) added to synthesize RNA.

    Article Title: A Simple and Efficient In Vivo Non-viral RNA Transfection Method for Labeling the Whole Axonal Tree of Individual Adult Long-Range Projection Neurons
    Article Snippet: After checking digestion by electrophoresis in 0.7% agarose gel, DNA templates were purified and precipitated. .. Finally, DNA template in vitro transcription was carried out using 1.5 μg of plasmidic DNA in a 50 μl distilled DEPC water solution containing 10× SP6 buffer (5 μl), rNTP mix (5 μl) (Amersham Pharmacia, UK), CAP analog (m7G(5′)ppp(5′)G) (5 μl) (New England Biolabs, Ipswich, MA, USA), Rnasin (1.5 μl) (Promega, Madison, WI, USA) and SP6 RNA polymerase (0.5 μl; New England Biolabs, Ipswich, MA, USA). .. After checking the synthesized Sindbis-Pal-eGFP RNA integrity on an electrophoresis gel ( Figure ), this stock RNA solution (1.8–2 μg/μl) was stored at -80°C.

    Article Title: HvCEBiP, a gene homologous to rice chitin receptor CEBiP, contributes to basal resistance of barley to Magnaporthe oryzae
    Article Snippet: BSMV genomic RNAs were transcribed in vitro as previously described with some modifications [ ]. .. The reaction was performed at 37°C for 60 min in 50 μl of reaction buffer containing 1 μg of linearized plasmids, 1 μl of T7 RNA polymerase (Takara), 10 μl of 50 mM DTT, 6 μl of 10 mM NTPs (rATP, rCTP, rUTP), 0.4 μl of 10 mM rGTP and 5 μl of 5 mM m7 G(ppp)G RNA cap structure analog (New England Biolabs).

    Article Title: RNAe: an effective method for targeted protein translation enhancement by artificial non-coding RNA with SINEB2 repeat
    Article Snippet: RNA without 5' cap for transfection was transcribed in vitro by HiScribe™ T7 High Yield RNA Synthesis Kit (New England Biolabs, UK). .. RNA with 5' cap was transcribed also by HiScribe™ T7 High Yield RNA Synthesis Kit with m7G(5′)ppp(5′)G (New England Biolabs, UK) added.

    Article Title: eIF3 targets cell proliferation mRNAs for translational activation or repression
    Article Snippet: Paragraph title: In vitro translation ... 1 mM m7 G(5′)ppp(5′)G RNA cap structure analog (NEB) was added to reactions when indicated.

    Article Title: Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA
    Article Snippet: Paragraph title: Generation of Poly-(A) Tailed DNA Fragments and In Vitro Transcribed mRNA ... We also used a Cap/NTP mixture with an m7G(5′)ppp(5′)G ARCA cap analog (New England Biolabs, Manchester, CT, USA), 3′-methylcytidine triphosphate and pseudo-uridine triphosphate (TriLink Biotechnologies, San Diego, CA, USA) following the protocol for generation of modified mRNA.

    Article Title: hnRNP A1 Interacts with the 5? Untranslated Regions of Enterovirus 71 and Sindbis Virus RNA and Is Required for Viral Replication
    Article Snippet: The positive-strand RNA was synthesized by in vitro transcription using T7 polymerase and labeled with [α-32 P]GTP. .. The RNA was capped by adding an m7G(5′)ppp(5′)G cap structural analog (NEB) (final concentration, 5 mM) into the reaction mixture and purified as described above.

    Spectrophotometry:

    Article Title: Characterization of Synthetic Chikungunya Viruses Based on the Consensus Sequence of Recent E1-226V Isolates
    Article Snippet: RNA was transcribed from plasmids with the phi2.5 promoter using the AmpliScribe T7 high yield transcription kit (Epicenter), the m7 GpppA RNA cap structure analog (NEB) and 0.7 µg of template DNA that had been linearized with Spe I. .. After a 3-h reaction at 37°C, template DNA was digested with DnaseI and RNA was purified by precipitation with 7.5 M LiCl (Ambion).

    Article Title: A Viral mRNA Motif at the 3′-Untranslated Region that Confers Translatability in a Cell-Specific Manner. Implications for Virus Evolution
    Article Snippet: Reactions containing 12.5 ng/μl of template DNA, 1000 U/ml of RNA polymerase, 1× RNAPol Reaction Buffer, 1 mM m7 G(5′)ppp(5′)G RNA Cap Structure Analog (New England Biolabs), 0.5 mM ATP, 0.5 mM CTP, 0.5 mM UTP, 0.25 mM GTP, and 0.32 U/μl RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen) were incubated for 4 h at 37 °C. .. The template DNA was then digested with 0.1 U/μl RNase-free Recombinant DNase I (Takara) for 10 minutes at 37 °C.

    Article Title: Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA
    Article Snippet: We also used a Cap/NTP mixture with an m7G(5′)ppp(5′)G ARCA cap analog (New England Biolabs, Manchester, CT, USA), 3′-methylcytidine triphosphate and pseudo-uridine triphosphate (TriLink Biotechnologies, San Diego, CA, USA) following the protocol for generation of modified mRNA. .. Reactions were incubated for 3–6 h at 37°C, and DNase and Antarctic phosphatase (New England Biolabs) were also added.

    Mobility Shift:

    Article Title: hnRNP A1 Interacts with the 5? Untranslated Regions of Enterovirus 71 and Sindbis Virus RNA and Is Required for Viral Replication
    Article Snippet: RNA probes for use in RNA gel mobility shift assays (EMSAs) were generated by runoff transcription using bacteriophage T7 RNA polymerase and then purified by use of an RNeasy minikit (Qiagen) and labeled at their 5′ ends by using T4 polynucleotide kinase and [γ-32 P]ATP. .. The RNA was capped by adding an m7G(5′)ppp(5′)G cap structural analog (NEB) (final concentration, 5 mM) into the reaction mixture and purified as described above.

    Migration:

    Article Title: hnRNP A1 Interacts with the 5? Untranslated Regions of Enterovirus 71 and Sindbis Virus RNA and Is Required for Viral Replication
    Article Snippet: The RNA was capped by adding an m7G(5′)ppp(5′)G cap structural analog (NEB) (final concentration, 5 mM) into the reaction mixture and purified as described above. .. The reaction was carried out in binding buffer (10 mM HEPES [pH 7.5], 150 mM KCl, 0.5 mM EGTA, 2 mM MgCl2 , 1 mM dithiothreitol, 1 unit RNasin,10% glycerol), and the final volume of the reaction mixture was 10 μl.

    Lysis:

    Article Title: eIF3 targets cell proliferation mRNAs for translational activation or repression
    Article Snippet: Cells were washed once with cold PBS (137 mM NaCl, 2.7 mM KCl, 100 mM Na2 HPO4 , 2 mM KH2 PO4 ) and an equal volume of freshly made cold lysis buffer (10 mM HEPES-KOH pH 7.6, 10 mM KOAc, 0.5 mM Mg(OAc)2 , 5 mM DTT, and 1 Complete EDTA-free Proteinase Inhibitor Cocktail tablet (Roche) per 10 ml of buffer) was added. .. 1 mM m7 G(5′)ppp(5′)G RNA cap structure analog (NEB) was added to reactions when indicated.

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    New England Biolabs m7 g
    M7 G, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m7 g/product/New England Biolabs
    Average 99 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    m7 g - by Bioz Stars, 2019-10
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    99
    New England Biolabs m7g cap analog
    Mammalian insulin receptor and insulin-like growth factor receptor 5′UTR provide resistance to eIF4a inhibition. ( A ) Diagram of RNAs used in the in vitro translation assays. ( B ) In vitro Translation in the absence (white bars) and presence (black bars) of excess <t>m7G</t> analogue. Data are plotted as the fraction activity of the mock treated extracts (error bars indicate SEM). ( C ) Titration of hippuristanol in in vitro translation assays. Data are plotted as the fraction activity of the mock treated extracts (error bars indicate SEM). DOI: http://dx.doi.org/10.7554/eLife.00542.007
    M7g Cap Analog, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m7g cap analog/product/New England Biolabs
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    m7g cap analog - by Bioz Stars, 2019-10
    99/100 stars
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    Mammalian insulin receptor and insulin-like growth factor receptor 5′UTR provide resistance to eIF4a inhibition. ( A ) Diagram of RNAs used in the in vitro translation assays. ( B ) In vitro Translation in the absence (white bars) and presence (black bars) of excess m7G analogue. Data are plotted as the fraction activity of the mock treated extracts (error bars indicate SEM). ( C ) Titration of hippuristanol in in vitro translation assays. Data are plotted as the fraction activity of the mock treated extracts (error bars indicate SEM). DOI: http://dx.doi.org/10.7554/eLife.00542.007

    Journal: eLife

    Article Title: The insulin receptor cellular IRES confers resistance to eIF4A inhibition

    doi: 10.7554/eLife.00542

    Figure Lengend Snippet: Mammalian insulin receptor and insulin-like growth factor receptor 5′UTR provide resistance to eIF4a inhibition. ( A ) Diagram of RNAs used in the in vitro translation assays. ( B ) In vitro Translation in the absence (white bars) and presence (black bars) of excess m7G analogue. Data are plotted as the fraction activity of the mock treated extracts (error bars indicate SEM). ( C ) Titration of hippuristanol in in vitro translation assays. Data are plotted as the fraction activity of the mock treated extracts (error bars indicate SEM). DOI: http://dx.doi.org/10.7554/eLife.00542.007

    Article Snippet: For assays containing excess m7G cap, cap structure analogue (New England Biolabs, #S1407S) was added to a final concentration of 1 mM.

    Techniques: Inhibition, In Vitro, Activity Assay, Titration

    The APP 5′-leader demonstrates IRES activity in a monocistronic context. Monocistronic Photinus reporter mRNA with the β-globin or APP 5′-leader downstream of either an m7G cap or ApppG cap were co-transfected with monocistronic m7G-capped humanized Renilla luciferase mRNA, to normalize for transfection efficiency, into C6 cells. Following a 7 h transfection into C6 cells, Photinus and Renilla luciferase activities were assayed.

    Journal: Nucleic Acids Research

    Article Title: Regulating amyloid precursor protein synthesis through an internal ribosomal entry site

    doi: 10.1093/nar/gkn792

    Figure Lengend Snippet: The APP 5′-leader demonstrates IRES activity in a monocistronic context. Monocistronic Photinus reporter mRNA with the β-globin or APP 5′-leader downstream of either an m7G cap or ApppG cap were co-transfected with monocistronic m7G-capped humanized Renilla luciferase mRNA, to normalize for transfection efficiency, into C6 cells. Following a 7 h transfection into C6 cells, Photinus and Renilla luciferase activities were assayed.

    Article Snippet: The 0.5 μg of RNA, 100 mM of KCl, 0.25 mM of Mg acetate, 312.5 μM methionine and increasing concentrations of m7G cap analog (New England Biolabs) were added to the reaction and incubated for 1 h at 30°C.

    Techniques: Activity Assay, Transfection, Luciferase