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  • 97
    Roche rnase free dnase i
    Analysis of transcript levels in 7 h germlings. A. parasiticus SU-1, AFS10, and Δ veA were grown on YES solid media and spores were collected at 5 days. Germlings were grown from fresh spores in GMS liquid medium for 7 h, frozen in liquid nitrogen, and stored at −80 °C. RNA was extracted from germlings using grinding and sonication as described in Methods. RT-PCR was performed on total RNA treated with <t>RNAse-free</t> <t>DNAse</t> I with primers specific for each gene ( Table 3 ). PCR products were separated on a 1% agarose gel by electrophoresis. Citrate synthase, a constitutively expressed gene, was used as a positive control. gDNA, genomic DNA.
    Rnase Free Dnase I, supplied by Roche, used in various techniques. Bioz Stars score: 97/100, based on 3790 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega rnase free dnase i
    T7RNAP-driven transcription assay after conjugation between E.coli and T7RNAP-mitochondria. E.coli S17-1 harboring pT7hpGFP (lanes 1 and 2) and pT7hpGFP+oriT (lanes 3 and 4), respectively, were used as the donors. All mating samples were treated with 200 µg/ml of ampicillin, 200 U of <t>DNase</t> I and 50 µg/ml of <t>RNase</t> A for 2 h at 37°C after 5 h mating in an agarose block. After isolating total RNA from the mating mixture, RT–PCR was carried out using GFP -specific primers (see Materials and Methods). Expected band size of the RT–PCR GFP products was 423 bp. Lane M, 1 kb plus DNA ladder (Life Technologies). Control lanes (lanes 1 and 3) in which reverse transcriptase (RT) was omitted are indicated by minus signs.
    Rnase Free Dnase I, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 3762 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim rnase free dnase i
    <t>RNase</t> protection assay comparing amounts of intracellular HIV-2 genomic mRNA and of virion genomic mRNA. RNAs were harvested from 2 × 10 6 COS-1 cells, and from pelleted virions from the cell supernatants, 48 h after electroporation of 10 μg of plasmid DNA. RNAs were treated with 20 U of RNase-free <t>DNase</t> I for 4 h at 37°C and analyzed as described in Materials and Methods. (a) Probe design and expected fragments. (b) Lane M, 32 P-labeled RNA markers in vitro transcribed from templates of known size. Plasmids electroporated were pE32 (wild-type full-length HIV-2; lanes A), pE32Δψ (lanes B), and pE41 lanes E. Lanes F, separate transfection of pE32 (wild type). Results for cellular (lane C) and virion RNA (lane D) controls from COS-1 cells electroporated with a plasmid expressing only the probe sequence in sense orientation from the SV40 promoter are also shown. Lane P, free probe minus RNase (10% of the amount added to other samples to avoid overloading autoradiogram); unmarked lane just left of P, 100% of free probe added to other samples plus RNase; lane G, untransfected COS-1 cell RNA control. The sense transcript controls in lanes C and D indicate that substantial amounts of cellular RNA were not nonspecifically pelleted but the small amount of RNA measured in the Δψ (B, virions) and pE41 (E, virions) virion samples may in part represent cosedimented 0.2-μm-pore-size-filterable RNA-containing subcellular fragments in addition to encapsidated RNA.
    Rnase Free Dnase I, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 645 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare rnase free dnase i
    cDNA synthesis and RT activities of wild-type and mutant viruses. (A) Quantitative analysis of viral cDNA synthesis by real-time PCR. The amount of early reverse-transcribed viral DNA present in CEM cells was measured 8 h after infection with the wild-type (WT) or mutant viruses (shaded bars). All viral stocks were treated with <t>RNase-free</t> <t>DNase</t> I to remove potential contamination by plasmid DNA. The culture medium alone was used in the mock infection (hatched bar), and equivalent amounts of the respective heat-inactivated viruses (solid bars) were used as negative controls for infection. For normalization of the DNA input of each sample, the human β-globin gene was amplified under identical PCR conditions. The results are expressed as the number of copies of HIV-1 cDNA detected per microgram of total DNA extracted from infected cells. The values shown represent the averages of two independent experiments. In each experiment, the standards and test samples were run in duplicate. (B) Particle-associated RT activities of wild-type and C130S-containing mutant viruses. After transient transfection of 293T cells, WT (shaded bar) and mutant (open bars) viruses were harvested, concentrated, lysed, and assayed for particle-associated RT activity as described in Materials and Methods. The result for each viral clone is shown as the mean ± standard error of three experiments using independently derived virus stocks.
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    Epicentre Biotechnologies rnase free dnase i
    cDNA synthesis and RT activities of wild-type and mutant viruses. (A) Quantitative analysis of viral cDNA synthesis by real-time PCR. The amount of early reverse-transcribed viral DNA present in CEM cells was measured 8 h after infection with the wild-type (WT) or mutant viruses (shaded bars). All viral stocks were treated with <t>RNase-free</t> <t>DNase</t> I to remove potential contamination by plasmid DNA. The culture medium alone was used in the mock infection (hatched bar), and equivalent amounts of the respective heat-inactivated viruses (solid bars) were used as negative controls for infection. For normalization of the DNA input of each sample, the human β-globin gene was amplified under identical PCR conditions. The results are expressed as the number of copies of HIV-1 cDNA detected per microgram of total DNA extracted from infected cells. The values shown represent the averages of two independent experiments. In each experiment, the standards and test samples were run in duplicate. (B) Particle-associated RT activities of wild-type and C130S-containing mutant viruses. After transient transfection of 293T cells, WT (shaded bar) and mutant (open bars) viruses were harvested, concentrated, lysed, and assayed for particle-associated RT activity as described in Materials and Methods. The result for each viral clone is shown as the mean ± standard error of three experiments using independently derived virus stocks.
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    FUJIFILM rnase free dnase i
    cDNA synthesis and RT activities of wild-type and mutant viruses. (A) Quantitative analysis of viral cDNA synthesis by real-time PCR. The amount of early reverse-transcribed viral DNA present in CEM cells was measured 8 h after infection with the wild-type (WT) or mutant viruses (shaded bars). All viral stocks were treated with <t>RNase-free</t> <t>DNase</t> I to remove potential contamination by plasmid DNA. The culture medium alone was used in the mock infection (hatched bar), and equivalent amounts of the respective heat-inactivated viruses (solid bars) were used as negative controls for infection. For normalization of the DNA input of each sample, the human β-globin gene was amplified under identical PCR conditions. The results are expressed as the number of copies of HIV-1 cDNA detected per microgram of total DNA extracted from infected cells. The values shown represent the averages of two independent experiments. In each experiment, the standards and test samples were run in duplicate. (B) Particle-associated RT activities of wild-type and C130S-containing mutant viruses. After transient transfection of 293T cells, WT (shaded bar) and mutant (open bars) viruses were harvested, concentrated, lysed, and assayed for particle-associated RT activity as described in Materials and Methods. The result for each viral clone is shown as the mean ± standard error of three experiments using independently derived virus stocks.
    Rnase Free Dnase I, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene rnase free dnase i
    RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ ( a ), MRB-ζ ( b ), and MR1-delζ ( c ) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of <t>RNase-free</t> <t>DNase</t> I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed
    Rnase Free Dnase I, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 127 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Omega Bio-tek rnase free dnase i
    RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ ( a ), MRB-ζ ( b ), and MR1-delζ ( c ) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of <t>RNase-free</t> <t>DNase</t> I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed
    Rnase Free Dnase I, supplied by Omega Bio-tek, used in various techniques. Bioz Stars score: 92/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co rnase free dnase i
    RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ ( a ), MRB-ζ ( b ), and MR1-delζ ( c ) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of <t>RNase-free</t> <t>DNase</t> I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed
    Rnase Free Dnase I, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 93/100, based on 181 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MdBio Inc rnase free dnase i
    RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ ( a ), MRB-ζ ( b ), and MR1-delζ ( c ) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of <t>RNase-free</t> <t>DNase</t> I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed
    Rnase Free Dnase I, supplied by MdBio Inc, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck & Co rnase free dnase i
    RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ ( a ), MRB-ζ ( b ), and MR1-delζ ( c ) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of <t>RNase-free</t> <t>DNase</t> I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed
    Rnase Free Dnase I, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MACHEREY NAGEL rnase free dnase i
    RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ ( a ), MRB-ζ ( b ), and MR1-delζ ( c ) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of <t>RNase-free</t> <t>DNase</t> I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed
    Rnase Free Dnase I, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio Basic Canada rnase free dnase i
    RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ ( a ), MRB-ζ ( b ), and MR1-delζ ( c ) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of <t>RNase-free</t> <t>DNase</t> I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed
    Rnase Free Dnase I, supplied by Bio Basic Canada, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies rnase free dnase i
    RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ ( a ), MRB-ζ ( b ), and MR1-delζ ( c ) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of <t>RNase-free</t> <t>DNase</t> I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed
    Rnase Free Dnase I, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 91/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Ingelheim rnase free dnase i
    RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ ( a ), MRB-ζ ( b ), and MR1-delζ ( c ) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of <t>RNase-free</t> <t>DNase</t> I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed
    Rnase Free Dnase I, supplied by Boehringer Ingelheim, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CinnaGen Co rnase free dnase i
    RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ ( a ), MRB-ζ ( b ), and MR1-delζ ( c ) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of <t>RNase-free</t> <t>DNase</t> I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed
    Rnase Free Dnase I, supplied by CinnaGen Co, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SinaClon BioScience rnase free dnase i
    RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ ( a ), MRB-ζ ( b ), and MR1-delζ ( c ) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of <t>RNase-free</t> <t>DNase</t> I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed
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    GenHunter Corporation rnase free dnase i
    RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ ( a ), MRB-ζ ( b ), and MR1-delζ ( c ) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of <t>RNase-free</t> <t>DNase</t> I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed
    Rnase Free Dnase I, supplied by GenHunter Corporation, used in various techniques. Bioz Stars score: 92/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson rnase free dnase i
    RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ ( a ), MRB-ζ ( b ), and MR1-delζ ( c ) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of <t>RNase-free</t> <t>DNase</t> I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed
    Rnase Free Dnase I, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Norgen Biotek rnase free dnase i kit
    RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ ( a ), MRB-ζ ( b ), and MR1-delζ ( c ) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of <t>RNase-free</t> <t>DNase</t> I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed
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    Promega rqi rnase free dnase i
    RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ ( a ), MRB-ζ ( b ), and MR1-delζ ( c ) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of <t>RNase-free</t> <t>DNase</t> I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed
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    TaKaRa rnase free dnase i kit
    RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ ( a ), MRB-ζ ( b ), and MR1-delζ ( c ) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of <t>RNase-free</t> <t>DNase</t> I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed
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    RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ ( a ), MRB-ζ ( b ), and MR1-delζ ( c ) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of <t>RNase-free</t> <t>DNase</t> I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed
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    Image Search Results


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

    Journal: Toxins

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

    doi: 10.3390/toxins7051411

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

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

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

    T7RNAP-driven transcription assay after conjugation between E.coli and T7RNAP-mitochondria. E.coli S17-1 harboring pT7hpGFP (lanes 1 and 2) and pT7hpGFP+oriT (lanes 3 and 4), respectively, were used as the donors. All mating samples were treated with 200 µg/ml of ampicillin, 200 U of DNase I and 50 µg/ml of RNase A for 2 h at 37°C after 5 h mating in an agarose block. After isolating total RNA from the mating mixture, RT–PCR was carried out using GFP -specific primers (see Materials and Methods). Expected band size of the RT–PCR GFP products was 423 bp. Lane M, 1 kb plus DNA ladder (Life Technologies). Control lanes (lanes 1 and 3) in which reverse transcriptase (RT) was omitted are indicated by minus signs.

    Journal: Nucleic Acids Research

    Article Title: Transformation of isolated mammalian mitochondria by bacterial conjugation

    doi: 10.1093/nar/gni140

    Figure Lengend Snippet: T7RNAP-driven transcription assay after conjugation between E.coli and T7RNAP-mitochondria. E.coli S17-1 harboring pT7hpGFP (lanes 1 and 2) and pT7hpGFP+oriT (lanes 3 and 4), respectively, were used as the donors. All mating samples were treated with 200 µg/ml of ampicillin, 200 U of DNase I and 50 µg/ml of RNase A for 2 h at 37°C after 5 h mating in an agarose block. After isolating total RNA from the mating mixture, RT–PCR was carried out using GFP -specific primers (see Materials and Methods). Expected band size of the RT–PCR GFP products was 423 bp. Lane M, 1 kb plus DNA ladder (Life Technologies). Control lanes (lanes 1 and 3) in which reverse transcriptase (RT) was omitted are indicated by minus signs.

    Article Snippet: RT–PCR analysis Total RNAs from the T7RNAP-mitochondria mating mixture or from electroporated mitochondria were collected by isopropyl alcohol precipitation, and residual DNA contaminants were removed using RNase-free DNase I (Promega, Madison, WI) as directed by the manufacturer.

    Techniques: Conjugation Assay, Blocking Assay, Reverse Transcription Polymerase Chain Reaction

    RNase protection assay comparing amounts of intracellular HIV-2 genomic mRNA and of virion genomic mRNA. RNAs were harvested from 2 × 10 6 COS-1 cells, and from pelleted virions from the cell supernatants, 48 h after electroporation of 10 μg of plasmid DNA. RNAs were treated with 20 U of RNase-free DNase I for 4 h at 37°C and analyzed as described in Materials and Methods. (a) Probe design and expected fragments. (b) Lane M, 32 P-labeled RNA markers in vitro transcribed from templates of known size. Plasmids electroporated were pE32 (wild-type full-length HIV-2; lanes A), pE32Δψ (lanes B), and pE41 lanes E. Lanes F, separate transfection of pE32 (wild type). Results for cellular (lane C) and virion RNA (lane D) controls from COS-1 cells electroporated with a plasmid expressing only the probe sequence in sense orientation from the SV40 promoter are also shown. Lane P, free probe minus RNase (10% of the amount added to other samples to avoid overloading autoradiogram); unmarked lane just left of P, 100% of free probe added to other samples plus RNase; lane G, untransfected COS-1 cell RNA control. The sense transcript controls in lanes C and D indicate that substantial amounts of cellular RNA were not nonspecifically pelleted but the small amount of RNA measured in the Δψ (B, virions) and pE41 (E, virions) virion samples may in part represent cosedimented 0.2-μm-pore-size-filterable RNA-containing subcellular fragments in addition to encapsidated RNA.

    Journal: Journal of Virology

    Article Title: Identification of a Human Immunodeficiency Virus Type 2 (HIV-2) Encapsidation Determinant and Transduction of Nondividing Human Cells by HIV-2-Based Lentivirus Vectors

    doi:

    Figure Lengend Snippet: RNase protection assay comparing amounts of intracellular HIV-2 genomic mRNA and of virion genomic mRNA. RNAs were harvested from 2 × 10 6 COS-1 cells, and from pelleted virions from the cell supernatants, 48 h after electroporation of 10 μg of plasmid DNA. RNAs were treated with 20 U of RNase-free DNase I for 4 h at 37°C and analyzed as described in Materials and Methods. (a) Probe design and expected fragments. (b) Lane M, 32 P-labeled RNA markers in vitro transcribed from templates of known size. Plasmids electroporated were pE32 (wild-type full-length HIV-2; lanes A), pE32Δψ (lanes B), and pE41 lanes E. Lanes F, separate transfection of pE32 (wild type). Results for cellular (lane C) and virion RNA (lane D) controls from COS-1 cells electroporated with a plasmid expressing only the probe sequence in sense orientation from the SV40 promoter are also shown. Lane P, free probe minus RNase (10% of the amount added to other samples to avoid overloading autoradiogram); unmarked lane just left of P, 100% of free probe added to other samples plus RNase; lane G, untransfected COS-1 cell RNA control. The sense transcript controls in lanes C and D indicate that substantial amounts of cellular RNA were not nonspecifically pelleted but the small amount of RNA measured in the Δψ (B, virions) and pE41 (E, virions) virion samples may in part represent cosedimented 0.2-μm-pore-size-filterable RNA-containing subcellular fragments in addition to encapsidated RNA.

    Article Snippet: DNase treatment of vectors was performed with 50 U of RNase-free DNase I (Boehringer Mannheim) per ml for 2 h at 37°C.

    Techniques: Rnase Protection Assay, Electroporation, Plasmid Preparation, Labeling, In Vitro, Transfection, Expressing, Sequencing

    cDNA synthesis and RT activities of wild-type and mutant viruses. (A) Quantitative analysis of viral cDNA synthesis by real-time PCR. The amount of early reverse-transcribed viral DNA present in CEM cells was measured 8 h after infection with the wild-type (WT) or mutant viruses (shaded bars). All viral stocks were treated with RNase-free DNase I to remove potential contamination by plasmid DNA. The culture medium alone was used in the mock infection (hatched bar), and equivalent amounts of the respective heat-inactivated viruses (solid bars) were used as negative controls for infection. For normalization of the DNA input of each sample, the human β-globin gene was amplified under identical PCR conditions. The results are expressed as the number of copies of HIV-1 cDNA detected per microgram of total DNA extracted from infected cells. The values shown represent the averages of two independent experiments. In each experiment, the standards and test samples were run in duplicate. (B) Particle-associated RT activities of wild-type and C130S-containing mutant viruses. After transient transfection of 293T cells, WT (shaded bar) and mutant (open bars) viruses were harvested, concentrated, lysed, and assayed for particle-associated RT activity as described in Materials and Methods. The result for each viral clone is shown as the mean ± standard error of three experiments using independently derived virus stocks.

    Journal: Journal of Virology

    Article Title: Requirement for Integrase during Reverse Transcription of Human Immunodeficiency Virus Type 1 and the Effect of Cysteine Mutations of Integrase on Its Interactions with Reverse Transcriptase

    doi: 10.1128/JVI.78.10.5045-5055.2004

    Figure Lengend Snippet: cDNA synthesis and RT activities of wild-type and mutant viruses. (A) Quantitative analysis of viral cDNA synthesis by real-time PCR. The amount of early reverse-transcribed viral DNA present in CEM cells was measured 8 h after infection with the wild-type (WT) or mutant viruses (shaded bars). All viral stocks were treated with RNase-free DNase I to remove potential contamination by plasmid DNA. The culture medium alone was used in the mock infection (hatched bar), and equivalent amounts of the respective heat-inactivated viruses (solid bars) were used as negative controls for infection. For normalization of the DNA input of each sample, the human β-globin gene was amplified under identical PCR conditions. The results are expressed as the number of copies of HIV-1 cDNA detected per microgram of total DNA extracted from infected cells. The values shown represent the averages of two independent experiments. In each experiment, the standards and test samples were run in duplicate. (B) Particle-associated RT activities of wild-type and C130S-containing mutant viruses. After transient transfection of 293T cells, WT (shaded bar) and mutant (open bars) viruses were harvested, concentrated, lysed, and assayed for particle-associated RT activity as described in Materials and Methods. The result for each viral clone is shown as the mean ± standard error of three experiments using independently derived virus stocks.

    Article Snippet: Fifty nanograms of p24 equivalent of viral stock was treated with 5 U of RNase-free DNase I (Amersham Pharmacia) at room temperature for 1 h. An equal amount of viral stock was heat inactivated by incubation at 65°C for 2 h after DNase I treatment.

    Techniques: Mutagenesis, Real-time Polymerase Chain Reaction, Infection, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Transfection, Activity Assay, Derivative Assay

    RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ ( a ), MRB-ζ ( b ), and MR1-delζ ( c ) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of RNase-free DNase I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed

    Journal: Journal of neuro-oncology

    Article Title: Genetically engineered T cells to target EGFRvIII expressing glioblastoma

    doi: 10.1007/s11060-009-9889-1

    Figure Lengend Snippet: RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ ( a ), MRB-ζ ( b ), and MR1-delζ ( c ) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of RNase-free DNase I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed

    Article Snippet: To eliminate residual contaminating DNA, 1 U of RNase-free DNase I (Stratagene, La Jolla, CA) was added to each sample during the RNA purification procedure.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, RNA Expression, Amplification, Agarose Gel Electrophoresis