monarch rna cleanup kit  (New England Biolabs)


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    New England Biolabs monarch rna cleanup kit
    rpoS mRNA decay curves across wild type and bosR mutant. Wild-type B. burgdorferi strain B31 (Wt) and the bosR mutant (Δ bosR ) were cultured in BSK-II <t>at</t> <t>37°C</t> to stationary phase. Transcriptional arrest was induced by adding actinomycin D (150 μg/ml) , and samples were collected at various time points after the actinomycin D treatment. RNAs were extracted and subjected to qRT-PCR analyses for quantification of the copy numbers of rpoS mRNA ( A ) or flaB mRNA ( B ). The fraction of remaining <t>RNA</t> ( f ) was calculated and plotted as log values. Closed circles and open triangles represent the remaining RNA fraction in wild-type B. burgdorferi and the bosR mutant, respectively. The error bars represent standard deviation of three independent experiments. * P < 0.01; ** P < 0.001, using t -test.
    Monarch Rna Cleanup Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "A Fur family protein BosR is a novel RNA-binding protein that controls rpoS RNA stability in the Lyme disease pathogen"

    Article Title: A Fur family protein BosR is a novel RNA-binding protein that controls rpoS RNA stability in the Lyme disease pathogen

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkae114

    rpoS mRNA decay curves across wild type and bosR mutant. Wild-type B. burgdorferi strain B31 (Wt) and the bosR mutant (Δ bosR ) were cultured in BSK-II at 37°C to stationary phase. Transcriptional arrest was induced by adding actinomycin D (150 μg/ml) , and samples were collected at various time points after the actinomycin D treatment. RNAs were extracted and subjected to qRT-PCR analyses for quantification of the copy numbers of rpoS mRNA ( A ) or flaB mRNA ( B ). The fraction of remaining RNA ( f ) was calculated and plotted as log values. Closed circles and open triangles represent the remaining RNA fraction in wild-type B. burgdorferi and the bosR mutant, respectively. The error bars represent standard deviation of three independent experiments. * P < 0.01; ** P < 0.001, using t -test.
    Figure Legend Snippet: rpoS mRNA decay curves across wild type and bosR mutant. Wild-type B. burgdorferi strain B31 (Wt) and the bosR mutant (Δ bosR ) were cultured in BSK-II at 37°C to stationary phase. Transcriptional arrest was induced by adding actinomycin D (150 μg/ml) , and samples were collected at various time points after the actinomycin D treatment. RNAs were extracted and subjected to qRT-PCR analyses for quantification of the copy numbers of rpoS mRNA ( A ) or flaB mRNA ( B ). The fraction of remaining RNA ( f ) was calculated and plotted as log values. Closed circles and open triangles represent the remaining RNA fraction in wild-type B. burgdorferi and the bosR mutant, respectively. The error bars represent standard deviation of three independent experiments. * P < 0.01; ** P < 0.001, using t -test.

    Techniques Used: Mutagenesis, Cell Culture, Quantitative RT-PCR, Standard Deviation

    RNA immunoprecipitation (RIP) demonstrates BosR binding to rpoS mRNA in cellulo . ( A ) IPTG induction of HA-tagged BosR. Wild-type B. burgdorferi strain B31 expressing a shuttle vector carrying lacp-bosR-HA was cultured in BSK-II at 37°C in the presence of various concentrations of IPTG. Spirochetes were harvested at stationary phase and subjected to SDS-PAGE (top panel) and immunoblotting analyses (lower panel). ( B ) RNA immunoprecipitation. Spirochetes grown in the presence of 100 μg/ml of IPTG were subjected to RIP using anti-HA, anti-YebC, or normal mouse IgG. Immunoprecipitated RNA samples were subjected to qRT-PCR analyses to determine the copy numbers of rpoS, ospC , rrp2 , and rpoN mRNAs (labeled on top). The mRNA levels for each gene in BosR-HA and YebC samples were normalized with the mRNA levels of the IgG sample for each gene (in which the value was set as 1.0). The bars represent the mean values of three independent experiments, and the error bars represent the standard deviation. **** P < 0.00001, using one-way ANOVA.
    Figure Legend Snippet: RNA immunoprecipitation (RIP) demonstrates BosR binding to rpoS mRNA in cellulo . ( A ) IPTG induction of HA-tagged BosR. Wild-type B. burgdorferi strain B31 expressing a shuttle vector carrying lacp-bosR-HA was cultured in BSK-II at 37°C in the presence of various concentrations of IPTG. Spirochetes were harvested at stationary phase and subjected to SDS-PAGE (top panel) and immunoblotting analyses (lower panel). ( B ) RNA immunoprecipitation. Spirochetes grown in the presence of 100 μg/ml of IPTG were subjected to RIP using anti-HA, anti-YebC, or normal mouse IgG. Immunoprecipitated RNA samples were subjected to qRT-PCR analyses to determine the copy numbers of rpoS, ospC , rrp2 , and rpoN mRNAs (labeled on top). The mRNA levels for each gene in BosR-HA and YebC samples were normalized with the mRNA levels of the IgG sample for each gene (in which the value was set as 1.0). The bars represent the mean values of three independent experiments, and the error bars represent the standard deviation. **** P < 0.00001, using one-way ANOVA.

    Techniques Used: Immunoprecipitation, Binding Assay, Expressing, Plasmid Preparation, Cell Culture, SDS Page, Western Blot, Quantitative RT-PCR, Labeling, Standard Deviation

    The role of 5′ UTR on rpoS expression. ( A ) Schematic representation showing the mutated 5′ UTR rpoS gene on the shuttle vector. The open box labeled with –24–12 represents the major rpoS promoter (a σ 54 -type promoter). The arrow indicates the transcription start site (TSS) of rpoS . Shine-Dalgarno sequence is represented in a square box labeled as SD. The ATG start codon of rpoS ORF is in bold. pSR080 harbors a native copy of rpoS with its minimal σ 54 -type promoter. pSR079 harbors a mutated version of rpoS in which the 5′UTR was replaced with a 50bp flaB UTR (indicated in Red). ( B ) RNA decay assays. The bosR mutant carrying either pSR079 or pSR080 was subjected to RNA decay assays as described in Figure . Open circles and closed triangles represent the remaining RNA fraction. The error bars represent the standard deviation of three independent experiments. *** P < 0.0001, using t -test. ( C ) The effect of 5′ UTR on RpoS production. Wild-type B. burgdorferi 5A14, the rpoS mutant (Δ rpoS ), the rpoS mutant containing pSR080 (Δ rpoS + pSR080) or pSR079 (Δ rpoS + pSR079), and the bosR mutant (Δ bosR ), the bosR mutant containing pSR080 (Δ bosR + pSR080) or pSR079 (Δ bosR + pSR079), were cultured in BSK-II medium at 37°C and harvested at the stationary phase. Cell lysates were subjected to SDS-PAGE (top panel) or immunoblot analyses (bottom panel). The bands corresponding to OspC, RpoS and FlaB were indicated on the right. ( D ) Quantitation of rpoS mRNA levels by qRT-PCR. RNAs were extracted from the cultures in (C) and subjected to qRT-PCR. The levels of rpoS mRNA in Δ rpoS + pSR080 were normalized as 1.0. The bars represent the mean values of three independent experiments, and the error bars represent the standard deviation. **** P < 0.00001; ** P < 0.001 using one-way ANOVA. ( E ) BosR-independent rpoS expression from pSR079 requires RpoN. Spirochetes were cultured as described in (C) and cell lysates were subjected to SDS-PAGE (top panel) or immunoblot analyses (bottom panel). Bands corresponding to RpoS, OspC and FlaB are indicated on the right.
    Figure Legend Snippet: The role of 5′ UTR on rpoS expression. ( A ) Schematic representation showing the mutated 5′ UTR rpoS gene on the shuttle vector. The open box labeled with –24–12 represents the major rpoS promoter (a σ 54 -type promoter). The arrow indicates the transcription start site (TSS) of rpoS . Shine-Dalgarno sequence is represented in a square box labeled as SD. The ATG start codon of rpoS ORF is in bold. pSR080 harbors a native copy of rpoS with its minimal σ 54 -type promoter. pSR079 harbors a mutated version of rpoS in which the 5′UTR was replaced with a 50bp flaB UTR (indicated in Red). ( B ) RNA decay assays. The bosR mutant carrying either pSR079 or pSR080 was subjected to RNA decay assays as described in Figure . Open circles and closed triangles represent the remaining RNA fraction. The error bars represent the standard deviation of three independent experiments. *** P < 0.0001, using t -test. ( C ) The effect of 5′ UTR on RpoS production. Wild-type B. burgdorferi 5A14, the rpoS mutant (Δ rpoS ), the rpoS mutant containing pSR080 (Δ rpoS + pSR080) or pSR079 (Δ rpoS + pSR079), and the bosR mutant (Δ bosR ), the bosR mutant containing pSR080 (Δ bosR + pSR080) or pSR079 (Δ bosR + pSR079), were cultured in BSK-II medium at 37°C and harvested at the stationary phase. Cell lysates were subjected to SDS-PAGE (top panel) or immunoblot analyses (bottom panel). The bands corresponding to OspC, RpoS and FlaB were indicated on the right. ( D ) Quantitation of rpoS mRNA levels by qRT-PCR. RNAs were extracted from the cultures in (C) and subjected to qRT-PCR. The levels of rpoS mRNA in Δ rpoS + pSR080 were normalized as 1.0. The bars represent the mean values of three independent experiments, and the error bars represent the standard deviation. **** P < 0.00001; ** P < 0.001 using one-way ANOVA. ( E ) BosR-independent rpoS expression from pSR079 requires RpoN. Spirochetes were cultured as described in (C) and cell lysates were subjected to SDS-PAGE (top panel) or immunoblot analyses (bottom panel). Bands corresponding to RpoS, OspC and FlaB are indicated on the right.

    Techniques Used: Expressing, Plasmid Preparation, Labeling, Sequencing, Mutagenesis, Standard Deviation, Cell Culture, SDS Page, Western Blot, Quantitation Assay, Quantitative RT-PCR

    monarch rna cleanup kit  (New England Biolabs)


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    New England Biolabs monarch rna cleanup kit
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    monarch rna cleanup kit  (New England Biolabs)


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    New England Biolabs monarch rna cleanup kit
    a Using <t>RNA</t> sequencing, a few significantly differentially expressed genes were detected in EBV-negative HK1 cells treated with mTZ3-LNP ( n = 3 experimental replicates). b Differentially expressed genes identified in SNU719 and C666-1 cells treated with mTZ3-LNP versus those treated with <t>control</t> <t>mRNA-LNP</t> for 48 h ( n = 3 experimental replicates). BZLF1 and EBV-encoded transcripts are illustrated in blue and red dots, respectively. c The viability of SNU719, C666-1, and HK1 cells treated with mTZ3-LNP alone and a combination of mTZ3-LNP and GCV for 24, 48, 72, and 96 h was determined ( n = 3 experimental replicates). Significant growth inhibition was observed in the EBV-positive SNU719 and C666-1 cells treated with mTZ3-LNP alone and in combination with GCV. Data are presented as mean ± SD. A P < 0.05 was considered to indicate statistical significance. One-way analysis of variance (ANOVA). Source data are provided as a file.
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    1) Product Images from "Synthetic BZLF1 -targeted transcriptional activator for efficient lytic induction therapy against EBV-associated epithelial cancers"

    Article Title: Synthetic BZLF1 -targeted transcriptional activator for efficient lytic induction therapy against EBV-associated epithelial cancers

    Journal: Nature Communications

    doi: 10.1038/s41467-024-48031-8

    a Using RNA sequencing, a few significantly differentially expressed genes were detected in EBV-negative HK1 cells treated with mTZ3-LNP ( n = 3 experimental replicates). b Differentially expressed genes identified in SNU719 and C666-1 cells treated with mTZ3-LNP versus those treated with control mRNA-LNP for 48 h ( n = 3 experimental replicates). BZLF1 and EBV-encoded transcripts are illustrated in blue and red dots, respectively. c The viability of SNU719, C666-1, and HK1 cells treated with mTZ3-LNP alone and a combination of mTZ3-LNP and GCV for 24, 48, 72, and 96 h was determined ( n = 3 experimental replicates). Significant growth inhibition was observed in the EBV-positive SNU719 and C666-1 cells treated with mTZ3-LNP alone and in combination with GCV. Data are presented as mean ± SD. A P < 0.05 was considered to indicate statistical significance. One-way analysis of variance (ANOVA). Source data are provided as a file.
    Figure Legend Snippet: a Using RNA sequencing, a few significantly differentially expressed genes were detected in EBV-negative HK1 cells treated with mTZ3-LNP ( n = 3 experimental replicates). b Differentially expressed genes identified in SNU719 and C666-1 cells treated with mTZ3-LNP versus those treated with control mRNA-LNP for 48 h ( n = 3 experimental replicates). BZLF1 and EBV-encoded transcripts are illustrated in blue and red dots, respectively. c The viability of SNU719, C666-1, and HK1 cells treated with mTZ3-LNP alone and a combination of mTZ3-LNP and GCV for 24, 48, 72, and 96 h was determined ( n = 3 experimental replicates). Significant growth inhibition was observed in the EBV-positive SNU719 and C666-1 cells treated with mTZ3-LNP alone and in combination with GCV. Data are presented as mean ± SD. A P < 0.05 was considered to indicate statistical significance. One-way analysis of variance (ANOVA). Source data are provided as a file.

    Techniques Used: RNA Sequencing Assay, Inhibition

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    New England Biolabs monarch rna cleanup kit
    rpoS mRNA decay curves across wild type and bosR mutant. Wild-type B. burgdorferi strain B31 (Wt) and the bosR mutant (Δ bosR ) were cultured in BSK-II <t>at</t> <t>37°C</t> to stationary phase. Transcriptional arrest was induced by adding actinomycin D (150 μg/ml) , and samples were collected at various time points after the actinomycin D treatment. RNAs were extracted and subjected to qRT-PCR analyses for quantification of the copy numbers of rpoS mRNA ( A ) or flaB mRNA ( B ). The fraction of remaining <t>RNA</t> ( f ) was calculated and plotted as log values. Closed circles and open triangles represent the remaining RNA fraction in wild-type B. burgdorferi and the bosR mutant, respectively. The error bars represent standard deviation of three independent experiments. * P < 0.01; ** P < 0.001, using t -test.
    Monarch Rna Cleanup Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rpoS mRNA decay curves across wild type and bosR mutant. Wild-type B. burgdorferi strain B31 (Wt) and the bosR mutant (Δ bosR ) were cultured in BSK-II at 37°C to stationary phase. Transcriptional arrest was induced by adding actinomycin D (150 μg/ml) , and samples were collected at various time points after the actinomycin D treatment. RNAs were extracted and subjected to qRT-PCR analyses for quantification of the copy numbers of rpoS mRNA ( A ) or flaB mRNA ( B ). The fraction of remaining RNA ( f ) was calculated and plotted as log values. Closed circles and open triangles represent the remaining RNA fraction in wild-type B. burgdorferi and the bosR mutant, respectively. The error bars represent standard deviation of three independent experiments. * P < 0.01; ** P < 0.001, using t -test.

    Journal: Nucleic Acids Research

    Article Title: A Fur family protein BosR is a novel RNA-binding protein that controls rpoS RNA stability in the Lyme disease pathogen

    doi: 10.1093/nar/gkae114

    Figure Lengend Snippet: rpoS mRNA decay curves across wild type and bosR mutant. Wild-type B. burgdorferi strain B31 (Wt) and the bosR mutant (Δ bosR ) were cultured in BSK-II at 37°C to stationary phase. Transcriptional arrest was induced by adding actinomycin D (150 μg/ml) , and samples were collected at various time points after the actinomycin D treatment. RNAs were extracted and subjected to qRT-PCR analyses for quantification of the copy numbers of rpoS mRNA ( A ) or flaB mRNA ( B ). The fraction of remaining RNA ( f ) was calculated and plotted as log values. Closed circles and open triangles represent the remaining RNA fraction in wild-type B. burgdorferi and the bosR mutant, respectively. The error bars represent standard deviation of three independent experiments. * P < 0.01; ** P < 0.001, using t -test.

    Article Snippet: Twenty-five μCi of α- 32 P UTP was added to the final reaction mixture and the in vitro transcription was achieved by incubating the mixture at 37°C for 4 h. RNA was purified using the Monarch® RNA Cleanup Kit (New England Biolabs).

    Techniques: Mutagenesis, Cell Culture, Quantitative RT-PCR, Standard Deviation

    RNA immunoprecipitation (RIP) demonstrates BosR binding to rpoS mRNA in cellulo . ( A ) IPTG induction of HA-tagged BosR. Wild-type B. burgdorferi strain B31 expressing a shuttle vector carrying lacp-bosR-HA was cultured in BSK-II at 37°C in the presence of various concentrations of IPTG. Spirochetes were harvested at stationary phase and subjected to SDS-PAGE (top panel) and immunoblotting analyses (lower panel). ( B ) RNA immunoprecipitation. Spirochetes grown in the presence of 100 μg/ml of IPTG were subjected to RIP using anti-HA, anti-YebC, or normal mouse IgG. Immunoprecipitated RNA samples were subjected to qRT-PCR analyses to determine the copy numbers of rpoS, ospC , rrp2 , and rpoN mRNAs (labeled on top). The mRNA levels for each gene in BosR-HA and YebC samples were normalized with the mRNA levels of the IgG sample for each gene (in which the value was set as 1.0). The bars represent the mean values of three independent experiments, and the error bars represent the standard deviation. **** P < 0.00001, using one-way ANOVA.

    Journal: Nucleic Acids Research

    Article Title: A Fur family protein BosR is a novel RNA-binding protein that controls rpoS RNA stability in the Lyme disease pathogen

    doi: 10.1093/nar/gkae114

    Figure Lengend Snippet: RNA immunoprecipitation (RIP) demonstrates BosR binding to rpoS mRNA in cellulo . ( A ) IPTG induction of HA-tagged BosR. Wild-type B. burgdorferi strain B31 expressing a shuttle vector carrying lacp-bosR-HA was cultured in BSK-II at 37°C in the presence of various concentrations of IPTG. Spirochetes were harvested at stationary phase and subjected to SDS-PAGE (top panel) and immunoblotting analyses (lower panel). ( B ) RNA immunoprecipitation. Spirochetes grown in the presence of 100 μg/ml of IPTG were subjected to RIP using anti-HA, anti-YebC, or normal mouse IgG. Immunoprecipitated RNA samples were subjected to qRT-PCR analyses to determine the copy numbers of rpoS, ospC , rrp2 , and rpoN mRNAs (labeled on top). The mRNA levels for each gene in BosR-HA and YebC samples were normalized with the mRNA levels of the IgG sample for each gene (in which the value was set as 1.0). The bars represent the mean values of three independent experiments, and the error bars represent the standard deviation. **** P < 0.00001, using one-way ANOVA.

    Article Snippet: Twenty-five μCi of α- 32 P UTP was added to the final reaction mixture and the in vitro transcription was achieved by incubating the mixture at 37°C for 4 h. RNA was purified using the Monarch® RNA Cleanup Kit (New England Biolabs).

    Techniques: Immunoprecipitation, Binding Assay, Expressing, Plasmid Preparation, Cell Culture, SDS Page, Western Blot, Quantitative RT-PCR, Labeling, Standard Deviation

    The role of 5′ UTR on rpoS expression. ( A ) Schematic representation showing the mutated 5′ UTR rpoS gene on the shuttle vector. The open box labeled with –24–12 represents the major rpoS promoter (a σ 54 -type promoter). The arrow indicates the transcription start site (TSS) of rpoS . Shine-Dalgarno sequence is represented in a square box labeled as SD. The ATG start codon of rpoS ORF is in bold. pSR080 harbors a native copy of rpoS with its minimal σ 54 -type promoter. pSR079 harbors a mutated version of rpoS in which the 5′UTR was replaced with a 50bp flaB UTR (indicated in Red). ( B ) RNA decay assays. The bosR mutant carrying either pSR079 or pSR080 was subjected to RNA decay assays as described in Figure . Open circles and closed triangles represent the remaining RNA fraction. The error bars represent the standard deviation of three independent experiments. *** P < 0.0001, using t -test. ( C ) The effect of 5′ UTR on RpoS production. Wild-type B. burgdorferi 5A14, the rpoS mutant (Δ rpoS ), the rpoS mutant containing pSR080 (Δ rpoS + pSR080) or pSR079 (Δ rpoS + pSR079), and the bosR mutant (Δ bosR ), the bosR mutant containing pSR080 (Δ bosR + pSR080) or pSR079 (Δ bosR + pSR079), were cultured in BSK-II medium at 37°C and harvested at the stationary phase. Cell lysates were subjected to SDS-PAGE (top panel) or immunoblot analyses (bottom panel). The bands corresponding to OspC, RpoS and FlaB were indicated on the right. ( D ) Quantitation of rpoS mRNA levels by qRT-PCR. RNAs were extracted from the cultures in (C) and subjected to qRT-PCR. The levels of rpoS mRNA in Δ rpoS + pSR080 were normalized as 1.0. The bars represent the mean values of three independent experiments, and the error bars represent the standard deviation. **** P < 0.00001; ** P < 0.001 using one-way ANOVA. ( E ) BosR-independent rpoS expression from pSR079 requires RpoN. Spirochetes were cultured as described in (C) and cell lysates were subjected to SDS-PAGE (top panel) or immunoblot analyses (bottom panel). Bands corresponding to RpoS, OspC and FlaB are indicated on the right.

    Journal: Nucleic Acids Research

    Article Title: A Fur family protein BosR is a novel RNA-binding protein that controls rpoS RNA stability in the Lyme disease pathogen

    doi: 10.1093/nar/gkae114

    Figure Lengend Snippet: The role of 5′ UTR on rpoS expression. ( A ) Schematic representation showing the mutated 5′ UTR rpoS gene on the shuttle vector. The open box labeled with –24–12 represents the major rpoS promoter (a σ 54 -type promoter). The arrow indicates the transcription start site (TSS) of rpoS . Shine-Dalgarno sequence is represented in a square box labeled as SD. The ATG start codon of rpoS ORF is in bold. pSR080 harbors a native copy of rpoS with its minimal σ 54 -type promoter. pSR079 harbors a mutated version of rpoS in which the 5′UTR was replaced with a 50bp flaB UTR (indicated in Red). ( B ) RNA decay assays. The bosR mutant carrying either pSR079 or pSR080 was subjected to RNA decay assays as described in Figure . Open circles and closed triangles represent the remaining RNA fraction. The error bars represent the standard deviation of three independent experiments. *** P < 0.0001, using t -test. ( C ) The effect of 5′ UTR on RpoS production. Wild-type B. burgdorferi 5A14, the rpoS mutant (Δ rpoS ), the rpoS mutant containing pSR080 (Δ rpoS + pSR080) or pSR079 (Δ rpoS + pSR079), and the bosR mutant (Δ bosR ), the bosR mutant containing pSR080 (Δ bosR + pSR080) or pSR079 (Δ bosR + pSR079), were cultured in BSK-II medium at 37°C and harvested at the stationary phase. Cell lysates were subjected to SDS-PAGE (top panel) or immunoblot analyses (bottom panel). The bands corresponding to OspC, RpoS and FlaB were indicated on the right. ( D ) Quantitation of rpoS mRNA levels by qRT-PCR. RNAs were extracted from the cultures in (C) and subjected to qRT-PCR. The levels of rpoS mRNA in Δ rpoS + pSR080 were normalized as 1.0. The bars represent the mean values of three independent experiments, and the error bars represent the standard deviation. **** P < 0.00001; ** P < 0.001 using one-way ANOVA. ( E ) BosR-independent rpoS expression from pSR079 requires RpoN. Spirochetes were cultured as described in (C) and cell lysates were subjected to SDS-PAGE (top panel) or immunoblot analyses (bottom panel). Bands corresponding to RpoS, OspC and FlaB are indicated on the right.

    Article Snippet: Twenty-five μCi of α- 32 P UTP was added to the final reaction mixture and the in vitro transcription was achieved by incubating the mixture at 37°C for 4 h. RNA was purified using the Monarch® RNA Cleanup Kit (New England Biolabs).

    Techniques: Expressing, Plasmid Preparation, Labeling, Sequencing, Mutagenesis, Standard Deviation, Cell Culture, SDS Page, Western Blot, Quantitation Assay, Quantitative RT-PCR