ssrna  (New England Biolabs)


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    ssRNA Ladder
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    ssRNA Ladder 25 gel lanes
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    New England Biolabs ssrna
    ssRNA Ladder
    ssRNA Ladder 25 gel lanes
    https://www.bioz.com/result/ssrna/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Images

    1) Product Images from "Viroplasm Protein P9-1 of Rice Black-Streaked Dwarf Virus Preferentially Binds to Single-Stranded RNA in Its Octamer Form, and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site"

    Article Title: Viroplasm Protein P9-1 of Rice Black-Streaked Dwarf Virus Preferentially Binds to Single-Stranded RNA in Its Octamer Form, and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site

    Journal: Journal of Virology

    doi: 10.1128/JVI.02264-13

    Competition and specificity assays of P9-1. (A) Increasing amounts of unlabeled competitor ssRNA, dsRNA, ssDNA, or dsDNA were mixed with 3.1 nmol DIG-labeled S9-1900nt ssRNA; 4.6 μmol of purified P9-1 was added to each sample, and the sample was
    Figure Legend Snippet: Competition and specificity assays of P9-1. (A) Increasing amounts of unlabeled competitor ssRNA, dsRNA, ssDNA, or dsDNA were mixed with 3.1 nmol DIG-labeled S9-1900nt ssRNA; 4.6 μmol of purified P9-1 was added to each sample, and the sample was

    Techniques Used: Labeling, Purification

    Formation of gradually shifted higher-order complexes by P9-1 and RNA. (A) Increasing amounts of P9-1 were incubated with 3.1 nmol of DIG-labeled RBSDV S9-1900nt ssRNA. The complexes were resolved by electrophoresis on a 1% agarose gel, and blots were
    Figure Legend Snippet: Formation of gradually shifted higher-order complexes by P9-1 and RNA. (A) Increasing amounts of P9-1 were incubated with 3.1 nmol of DIG-labeled RBSDV S9-1900nt ssRNA. The complexes were resolved by electrophoresis on a 1% agarose gel, and blots were

    Techniques Used: Incubation, Labeling, Electrophoresis, Agarose Gel Electrophoresis

    2) Product Images from "Structure-function analysis of the RNA helicase maleless"

    Article Title: Structure-function analysis of the RNA helicase maleless

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm1108

    Predicted RNA-binding domains in MLE. ( A ) Schematic representation of the predicted features of MLE and of the expressed fragments containing individual domains that were analyzed for RNA-binding. All proteins were tagged, either with a C-terminal flag-tag or an N-terminal His 6 -tag (black bars). The numbers represent the MLE aa that delineate the domains. Insert: The purified recombinant MLE derivatives were separated by polyacrylamide gel electrophoresis (PAGE) and stained with Coomassie Blue. Lane M displays a size marker with indicated molecular weights in kDa. ( B ) Scheme of the ssRNA and dsRNA substrates. The sequence of roX2 is depicted in black, Sp6 promoter and T7 promoter are shown in grey. ( C ) Electrophoretic mobility shift assay (EMSA). The indicated amounts of the predicted RNA-binding domains (1–4 pmoles/50–200 nM) were incubated on ice in the presence of 25 fmol (1.25 nM) radiolabeled dsRNA or ssRNA. RNA–protein complexes (bracketed regions) were resolved on a 1.8% agarose gel and visualized by PhosphoImager analysis of the dried gel. Positions of ssRNA and dsRNA are indicated. ( D ) Filter-binding assay. Binding of the predicted RNA-binding domains to dsRNA was analyzed by filter-binding as described in Materials and Methods section. Each sample was analyzed in duplicates and error bars reflect the experimental variation.
    Figure Legend Snippet: Predicted RNA-binding domains in MLE. ( A ) Schematic representation of the predicted features of MLE and of the expressed fragments containing individual domains that were analyzed for RNA-binding. All proteins were tagged, either with a C-terminal flag-tag or an N-terminal His 6 -tag (black bars). The numbers represent the MLE aa that delineate the domains. Insert: The purified recombinant MLE derivatives were separated by polyacrylamide gel electrophoresis (PAGE) and stained with Coomassie Blue. Lane M displays a size marker with indicated molecular weights in kDa. ( B ) Scheme of the ssRNA and dsRNA substrates. The sequence of roX2 is depicted in black, Sp6 promoter and T7 promoter are shown in grey. ( C ) Electrophoretic mobility shift assay (EMSA). The indicated amounts of the predicted RNA-binding domains (1–4 pmoles/50–200 nM) were incubated on ice in the presence of 25 fmol (1.25 nM) radiolabeled dsRNA or ssRNA. RNA–protein complexes (bracketed regions) were resolved on a 1.8% agarose gel and visualized by PhosphoImager analysis of the dried gel. Positions of ssRNA and dsRNA are indicated. ( D ) Filter-binding assay. Binding of the predicted RNA-binding domains to dsRNA was analyzed by filter-binding as described in Materials and Methods section. Each sample was analyzed in duplicates and error bars reflect the experimental variation.

    Techniques Used: RNA Binding Assay, FLAG-tag, Purification, Recombinant, Polyacrylamide Gel Electrophoresis, Staining, Marker, Sequencing, Electrophoretic Mobility Shift Assay, Incubation, Agarose Gel Electrophoresis, Filter-binding Assay, Binding Assay

    Stimulation of MLE ATPase activity by RNA. MLE and selected derivatives were analyzed in ATPase assays. 25 fmol (1.25 nM) of the indicated protein and increasing amounts of dsRNA (upper panels) or ssRNA (lower panels) were used in the ATPase reaction as described in Materials and Methods section. In ( B ) different enzyme preparations from ( A ) were used, with slightly different specific activities.
    Figure Legend Snippet: Stimulation of MLE ATPase activity by RNA. MLE and selected derivatives were analyzed in ATPase assays. 25 fmol (1.25 nM) of the indicated protein and increasing amounts of dsRNA (upper panels) or ssRNA (lower panels) were used in the ATPase reaction as described in Materials and Methods section. In ( B ) different enzyme preparations from ( A ) were used, with slightly different specific activities.

    Techniques Used: Activity Assay

    Contributions of MLE domains to helicase activity. Helicase reactions were carried out with increasing amounts (1.5, 3, 6, 12 fmole/75, 150, 300, 600 pM) of MLE and derivatives and 25 fmoles (1.25 nM) of radiolabeled dsRNA as described in Materials and Methods section. Lanes 1 and 2 indicate the migration position of the ssRNA (boiled substrate) and dsRNA substrate, respectively. Lanes 3–5 correspond to unwinding reactions from which ATP was omitted. In ( B ) different enzyme preparations from the ones tested in ( A ) were used, with enzyme inputs of 6, 12, 25 fmoles.
    Figure Legend Snippet: Contributions of MLE domains to helicase activity. Helicase reactions were carried out with increasing amounts (1.5, 3, 6, 12 fmole/75, 150, 300, 600 pM) of MLE and derivatives and 25 fmoles (1.25 nM) of radiolabeled dsRNA as described in Materials and Methods section. Lanes 1 and 2 indicate the migration position of the ssRNA (boiled substrate) and dsRNA substrate, respectively. Lanes 3–5 correspond to unwinding reactions from which ATP was omitted. In ( B ) different enzyme preparations from the ones tested in ( A ) were used, with enzyme inputs of 6, 12, 25 fmoles.

    Techniques Used: Activity Assay, Migration

    Binding of MLE and MLE derivatives to dsRNA and ssRNA. ( A ) Schematic representation of MLE deletion constructs analyzed in this study as in Figure 1 A. The insert to the right shows the purified recombinant MLE derivatives, separated by PAGE and stained with Coomassie Blue. ( B ) EMSA as in Figure 1 C with protein–RNA complexes (bracketed regions) resolved on a 1.8% agarose gel. Protein amounts range from 6 fmoles (0.3 nM) to 50 fmoles (2.5 nM). ( C ) dsRNA interactions measured by filter-binding as in Figure 1 D.
    Figure Legend Snippet: Binding of MLE and MLE derivatives to dsRNA and ssRNA. ( A ) Schematic representation of MLE deletion constructs analyzed in this study as in Figure 1 A. The insert to the right shows the purified recombinant MLE derivatives, separated by PAGE and stained with Coomassie Blue. ( B ) EMSA as in Figure 1 C with protein–RNA complexes (bracketed regions) resolved on a 1.8% agarose gel. Protein amounts range from 6 fmoles (0.3 nM) to 50 fmoles (2.5 nM). ( C ) dsRNA interactions measured by filter-binding as in Figure 1 D.

    Techniques Used: Binding Assay, Construct, Purification, Recombinant, Polyacrylamide Gel Electrophoresis, Staining, Agarose Gel Electrophoresis

    3) Product Images from "Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry"

    Article Title: Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry

    Journal: Journal of Chromatography. a

    doi: 10.1016/j.chroma.2016.12.062

    IP RP HPLC analyses of dsRNA and ssRNA under denaturing and non-denaturing conditions. (a) IP RP HPLC chromatogram of dsRNA at 50 °C. (b) IP RP HPLC chromatogram of dsRNA at 75 °C. (c) IP RP HPLC chromatogram of ssRNA at 50 °C. (d) IP RP HPLC chromatogram of ssRNA at 75 °C. (e) IP RP HPLC chromatogram of in vitro transcribed dsRNA with an excess of ssRNA. (f) IP RP HPLC chromatogram of purified dsRNA. Following in vitro transcription, the RNA was purified in a single step using RNase T1/DNase in conjunction with solid phase extraction. 2–3 μg of in vitro transcribed ds/ssRNA was analysed using gradient 1 at 260 nm UV detection.
    Figure Legend Snippet: IP RP HPLC analyses of dsRNA and ssRNA under denaturing and non-denaturing conditions. (a) IP RP HPLC chromatogram of dsRNA at 50 °C. (b) IP RP HPLC chromatogram of dsRNA at 75 °C. (c) IP RP HPLC chromatogram of ssRNA at 50 °C. (d) IP RP HPLC chromatogram of ssRNA at 75 °C. (e) IP RP HPLC chromatogram of in vitro transcribed dsRNA with an excess of ssRNA. (f) IP RP HPLC chromatogram of purified dsRNA. Following in vitro transcription, the RNA was purified in a single step using RNase T1/DNase in conjunction with solid phase extraction. 2–3 μg of in vitro transcribed ds/ssRNA was analysed using gradient 1 at 260 nm UV detection.

    Techniques Used: High Performance Liquid Chromatography, In Vitro, Purification

    Purification of dsRNA from E. coli . (a) Agarose gel electrophoresis of purified in vitro transcribed dsRNA and ssRNA. Following in vitro transcription, 2 μg of dsRNA containing excess ssRNA were incubated with RNase T1 in both the presence and absence of 0.5 M NaCl and purified by SPE. (b) Agarose gel electrophoresis of extracted and purified dsRNA. Following TRIzol extraction of total RNA from E. coli expressing dsRNA samples were incubated with RNase T1 in both the presence and absence of 0.3 M NaCl as indicated prior to purification using solid phase extraction. Control experiments were performed using total RNA from non-induced E. coli . (c) IP RP HPLC chromatogram of purified dsRNA. Following TRIzol extraction the total RNA from E. coli was purified in a single step using RNase T1/DNase in conjunction with solid phase extraction prior to analysis using IP RP HPLC gradient condition 1 at 260 nm UV detection.
    Figure Legend Snippet: Purification of dsRNA from E. coli . (a) Agarose gel electrophoresis of purified in vitro transcribed dsRNA and ssRNA. Following in vitro transcription, 2 μg of dsRNA containing excess ssRNA were incubated with RNase T1 in both the presence and absence of 0.5 M NaCl and purified by SPE. (b) Agarose gel electrophoresis of extracted and purified dsRNA. Following TRIzol extraction of total RNA from E. coli expressing dsRNA samples were incubated with RNase T1 in both the presence and absence of 0.3 M NaCl as indicated prior to purification using solid phase extraction. Control experiments were performed using total RNA from non-induced E. coli . (c) IP RP HPLC chromatogram of purified dsRNA. Following TRIzol extraction the total RNA from E. coli was purified in a single step using RNase T1/DNase in conjunction with solid phase extraction prior to analysis using IP RP HPLC gradient condition 1 at 260 nm UV detection.

    Techniques Used: Purification, Agarose Gel Electrophoresis, In Vitro, Incubation, Expressing, High Performance Liquid Chromatography

    4) Product Images from "Viroplasm Protein P9-1 of Rice Black-Streaked Dwarf Virus Preferentially Binds to Single-Stranded RNA in Its Octamer Form, and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site"

    Article Title: Viroplasm Protein P9-1 of Rice Black-Streaked Dwarf Virus Preferentially Binds to Single-Stranded RNA in Its Octamer Form, and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site

    Journal: Journal of Virology

    doi: 10.1128/JVI.02264-13

    Competition and specificity assays of P9-1. (A) Increasing amounts of unlabeled competitor ssRNA, dsRNA, ssDNA, or dsDNA were mixed with 3.1 nmol DIG-labeled S9-1900nt ssRNA; 4.6 μmol of purified P9-1 was added to each sample, and the sample was
    Figure Legend Snippet: Competition and specificity assays of P9-1. (A) Increasing amounts of unlabeled competitor ssRNA, dsRNA, ssDNA, or dsDNA were mixed with 3.1 nmol DIG-labeled S9-1900nt ssRNA; 4.6 μmol of purified P9-1 was added to each sample, and the sample was

    Techniques Used: Labeling, Purification

    Formation of gradually shifted higher-order complexes by P9-1 and RNA. (A) Increasing amounts of P9-1 were incubated with 3.1 nmol of DIG-labeled RBSDV S9-1900nt ssRNA. The complexes were resolved by electrophoresis on a 1% agarose gel, and blots were
    Figure Legend Snippet: Formation of gradually shifted higher-order complexes by P9-1 and RNA. (A) Increasing amounts of P9-1 were incubated with 3.1 nmol of DIG-labeled RBSDV S9-1900nt ssRNA. The complexes were resolved by electrophoresis on a 1% agarose gel, and blots were

    Techniques Used: Incubation, Labeling, Electrophoresis, Agarose Gel Electrophoresis

    5) Product Images from "2.7 Å cryo-EM structure of rotavirus core protein VP3, a unique capping machine with a helicase activity"

    Article Title: 2.7 Å cryo-EM structure of rotavirus core protein VP3, a unique capping machine with a helicase activity

    Journal: Science Advances

    doi: 10.1126/sciadv.aay6410

    VP3 exhibits RTPase and RNA helicase activity. ( A ) An autoradiograph showing a VP3 or PDE domain concentration-dependent decrease in the 5′-end [γ- 32 P] GTP-labeled RNA band intensity confirms that VP3 exhibits RTPase activity and that this activity is associated with the PDE domain. RV NSP2, which was previously shown to have RTPase activity, is used as a positive control. The RTPase activity of VP3 is inhibited in the presence of EDTA indicating that the RTPase activity of VP3 is metal dependent. ( B ) Low-resolution cryo-EM single-particle reconstruction of VP3 in complex with 8-mer single-stranded RNA (ssRNA), suggests that the RNA binds near a cleft in the PDE domain (black arrow). The difference map was obtained using low-resolution cryo-EM map from apo-VP3 and cryo-EM map from VP3-ssRNA complex in Chimera. ( C and D ) Biolayer interferometry (BLI) analysis of RV VP3 and PDE domain binding with ssRNA (8-mer) shows that both full-length VP3 and PDE domain bind with ssRNA with similar nanomolar affinity. The two–binding site model fits the data with excellent R 2 (~0.99) and χ 2 (~0) values. ( E and F ) Fluorescence-based helicase assay. The 6-fluorescin amidite (6-FAM)–labeled RNA strand is quenched by a complementary quencher [Black Hole Quencher (BHQ)–labeled] strand in dsRNA. Increased fluorescence intensity with increasing concentrations of VP3 indicates the strand separation confirming the dsRNA helicase activity of VP3. Helicase activity requires the full-length VP3; the PDE domain alone does not show any significant increase in fluorescence intensity. (F) The fluorescence intensity is also modulated by adenosine triphosphate (ATP) concentrations.
    Figure Legend Snippet: VP3 exhibits RTPase and RNA helicase activity. ( A ) An autoradiograph showing a VP3 or PDE domain concentration-dependent decrease in the 5′-end [γ- 32 P] GTP-labeled RNA band intensity confirms that VP3 exhibits RTPase activity and that this activity is associated with the PDE domain. RV NSP2, which was previously shown to have RTPase activity, is used as a positive control. The RTPase activity of VP3 is inhibited in the presence of EDTA indicating that the RTPase activity of VP3 is metal dependent. ( B ) Low-resolution cryo-EM single-particle reconstruction of VP3 in complex with 8-mer single-stranded RNA (ssRNA), suggests that the RNA binds near a cleft in the PDE domain (black arrow). The difference map was obtained using low-resolution cryo-EM map from apo-VP3 and cryo-EM map from VP3-ssRNA complex in Chimera. ( C and D ) Biolayer interferometry (BLI) analysis of RV VP3 and PDE domain binding with ssRNA (8-mer) shows that both full-length VP3 and PDE domain bind with ssRNA with similar nanomolar affinity. The two–binding site model fits the data with excellent R 2 (~0.99) and χ 2 (~0) values. ( E and F ) Fluorescence-based helicase assay. The 6-fluorescin amidite (6-FAM)–labeled RNA strand is quenched by a complementary quencher [Black Hole Quencher (BHQ)–labeled] strand in dsRNA. Increased fluorescence intensity with increasing concentrations of VP3 indicates the strand separation confirming the dsRNA helicase activity of VP3. Helicase activity requires the full-length VP3; the PDE domain alone does not show any significant increase in fluorescence intensity. (F) The fluorescence intensity is also modulated by adenosine triphosphate (ATP) concentrations.

    Techniques Used: Activity Assay, Autoradiography, Concentration Assay, Labeling, Positive Control, Binding Assay, Fluorescence, Helicase Assay

    Related Articles

    Marker:

    Article Title: Biochemical analysis of the Cas6-1 RNA endonuclease associated with the subtype I-D CRISPR-Cas system in Synechocystis sp. PCC 6803
    Article Snippet: To test whether the catalytic activity of the Cas6-1 H51A mutant can be restored, the cleavage buffer was supplemented with 500 mM imidazole, pH 8.0 and either pre-incubated at 4°C overnight or directly transferred to 30°C. .. The low range ssRNA ladder (ssRNA marker) (New England Biolabs), the RiboRuler low range RNA ladder (Thermo Fisher Scientific) and the microRNA marker (New England Biolabs) served as RNA ladders for size estimation. .. The CRISPR1 repeat oligo ladder (C1L) was generated by alkaline hydrolysis (50 mM Tris-HCl pH 8.5, 20 mM MgCl2 ) with 25 pmol substrate and was incubated for 72 h to 96 h at 30°C.

    In Vitro:

    Article Title: 2.7 Å cryo-EM structure of rotavirus core protein VP3, a unique capping machine with a helicase activity
    Article Snippet: For this purpose, first we synthesized RV gene 10 transcript using an in vitro transcription kit (Ambion T7 MEGAscript). .. GTase reaction mixture (20 μl) was prepared by adding 2 μl of IVT (in vitro transcription) synthesized ssRNA (200 ng/μl) with increasing concentrations of VP3 protein in GTase buffer [20 mM Hepes (pH 8.0), 150 mM NaCl, 5 mM DTT, 5 mM MgCl2 , and 5% glycerol], 2 μl of [α-32 P] GTP, and 1 μl of murine RNase inhibitor (New England Biolabs Inc.) with or without SAM (2 mM), followed by incubation at 30°C for 3 hours. .. The reaction was stopped by incubating the reaction mixture at 70°C for 5 min and adding 2× RNA gel loading buffer II (Thermo Fisher Scientific).

    Article Title: Viroplasm Protein P9-1 of Rice Black-Streaked Dwarf Virus Preferentially Binds to Single-Stranded RNA in Its Octamer Form, and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site
    Article Snippet: For one set of reactions, P9-1 was combined with DIG-labeled RNA probe and incubated for 10 min as described earlier, after which the competitor was added and the samples were incubated for an additional 10 min. .. The competitors used for competition between ssRNA and dsRNA and between ssDNA and dsDNA were as follows: ssRNA, full-length in vitro -transcribed S9 RNA transcript; dsRNA, annealing product of the plus and minus strands of the full-length S9 RNA; ssDNA, M13mp18 phage (NEB, Beijing, China); dsDNA, PCR amplification product of full-length RBSDV S9. ..

    Article Title: Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry
    Article Snippet: .. The following PCR parameters were used: the initial denaturation was 1 cycle of 30 s at 98 °C, 30 cycles of 30 s at 98 °C, 30 s at 68 °C, and 30 s at 72 °C and a final extension at 72 °C for 2 mins. dsRNA and ssRNA were also generated using in vitro transcription in conjunction with HiScribe™ T7 High Yield RNA Synthesis Kit (New England Biolabs): 10 mM NTPs, 1 x reaction buffer, 1 μg DNA template and 2 μL HiScribe™ T7 polymerase in 20 μL RNase-free water. .. 2.3 Expression of dsRNA gene using E. coli HT115(DE3) The E. coli strain, HT115(DE3) was obtained from Cold Spring Harbor Laboratory, NY, USA.

    Synthesized:

    Article Title: 2.7 Å cryo-EM structure of rotavirus core protein VP3, a unique capping machine with a helicase activity
    Article Snippet: For this purpose, first we synthesized RV gene 10 transcript using an in vitro transcription kit (Ambion T7 MEGAscript). .. GTase reaction mixture (20 μl) was prepared by adding 2 μl of IVT (in vitro transcription) synthesized ssRNA (200 ng/μl) with increasing concentrations of VP3 protein in GTase buffer [20 mM Hepes (pH 8.0), 150 mM NaCl, 5 mM DTT, 5 mM MgCl2 , and 5% glycerol], 2 μl of [α-32 P] GTP, and 1 μl of murine RNase inhibitor (New England Biolabs Inc.) with or without SAM (2 mM), followed by incubation at 30°C for 3 hours. .. The reaction was stopped by incubating the reaction mixture at 70°C for 5 min and adding 2× RNA gel loading buffer II (Thermo Fisher Scientific).

    Incubation:

    Article Title: 2.7 Å cryo-EM structure of rotavirus core protein VP3, a unique capping machine with a helicase activity
    Article Snippet: For this purpose, first we synthesized RV gene 10 transcript using an in vitro transcription kit (Ambion T7 MEGAscript). .. GTase reaction mixture (20 μl) was prepared by adding 2 μl of IVT (in vitro transcription) synthesized ssRNA (200 ng/μl) with increasing concentrations of VP3 protein in GTase buffer [20 mM Hepes (pH 8.0), 150 mM NaCl, 5 mM DTT, 5 mM MgCl2 , and 5% glycerol], 2 μl of [α-32 P] GTP, and 1 μl of murine RNase inhibitor (New England Biolabs Inc.) with or without SAM (2 mM), followed by incubation at 30°C for 3 hours. .. The reaction was stopped by incubating the reaction mixture at 70°C for 5 min and adding 2× RNA gel loading buffer II (Thermo Fisher Scientific).

    Electrophoretic Mobility Shift Assay:

    Article Title: Structure-function analysis of the RNA helicase maleless
    Article Snippet: Aliquots (10 µl) of each reaction were loaded onto a 10% polyacrylamide (30:1) gel in TBE and electrophoresed at 20 mA for 2–3 h. RNA was visualized by autoradiography of the dried gel. .. Electrophoretic mobility shift assay RNA-binding reaction (20 µl) contained radiolabeled ssRNA or dsRNA (25 fmol/1.25 nM) in Band-shift Buffer (20 mM Hepes-KOH pH 7.6, 3 mM MgCl2 , 10% glycerol, 1 mM DTT, 0.1 mg/ml BSA (NEB)). ..

    RNA Binding Assay:

    Article Title: Structure-function analysis of the RNA helicase maleless
    Article Snippet: Aliquots (10 µl) of each reaction were loaded onto a 10% polyacrylamide (30:1) gel in TBE and electrophoresed at 20 mA for 2–3 h. RNA was visualized by autoradiography of the dried gel. .. Electrophoretic mobility shift assay RNA-binding reaction (20 µl) contained radiolabeled ssRNA or dsRNA (25 fmol/1.25 nM) in Band-shift Buffer (20 mM Hepes-KOH pH 7.6, 3 mM MgCl2 , 10% glycerol, 1 mM DTT, 0.1 mg/ml BSA (NEB)). ..

    Polymerase Chain Reaction:

    Article Title: Viroplasm Protein P9-1 of Rice Black-Streaked Dwarf Virus Preferentially Binds to Single-Stranded RNA in Its Octamer Form, and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site
    Article Snippet: For one set of reactions, P9-1 was combined with DIG-labeled RNA probe and incubated for 10 min as described earlier, after which the competitor was added and the samples were incubated for an additional 10 min. .. The competitors used for competition between ssRNA and dsRNA and between ssDNA and dsDNA were as follows: ssRNA, full-length in vitro -transcribed S9 RNA transcript; dsRNA, annealing product of the plus and minus strands of the full-length S9 RNA; ssDNA, M13mp18 phage (NEB, Beijing, China); dsDNA, PCR amplification product of full-length RBSDV S9. ..

    Article Title: Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry
    Article Snippet: .. The following PCR parameters were used: the initial denaturation was 1 cycle of 30 s at 98 °C, 30 cycles of 30 s at 98 °C, 30 s at 68 °C, and 30 s at 72 °C and a final extension at 72 °C for 2 mins. dsRNA and ssRNA were also generated using in vitro transcription in conjunction with HiScribe™ T7 High Yield RNA Synthesis Kit (New England Biolabs): 10 mM NTPs, 1 x reaction buffer, 1 μg DNA template and 2 μL HiScribe™ T7 polymerase in 20 μL RNase-free water. .. 2.3 Expression of dsRNA gene using E. coli HT115(DE3) The E. coli strain, HT115(DE3) was obtained from Cold Spring Harbor Laboratory, NY, USA.

    Amplification:

    Article Title: Viroplasm Protein P9-1 of Rice Black-Streaked Dwarf Virus Preferentially Binds to Single-Stranded RNA in Its Octamer Form, and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site
    Article Snippet: For one set of reactions, P9-1 was combined with DIG-labeled RNA probe and incubated for 10 min as described earlier, after which the competitor was added and the samples were incubated for an additional 10 min. .. The competitors used for competition between ssRNA and dsRNA and between ssDNA and dsDNA were as follows: ssRNA, full-length in vitro -transcribed S9 RNA transcript; dsRNA, annealing product of the plus and minus strands of the full-length S9 RNA; ssDNA, M13mp18 phage (NEB, Beijing, China); dsDNA, PCR amplification product of full-length RBSDV S9. ..

    Generated:

    Article Title: Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry
    Article Snippet: .. The following PCR parameters were used: the initial denaturation was 1 cycle of 30 s at 98 °C, 30 cycles of 30 s at 98 °C, 30 s at 68 °C, and 30 s at 72 °C and a final extension at 72 °C for 2 mins. dsRNA and ssRNA were also generated using in vitro transcription in conjunction with HiScribe™ T7 High Yield RNA Synthesis Kit (New England Biolabs): 10 mM NTPs, 1 x reaction buffer, 1 μg DNA template and 2 μL HiScribe™ T7 polymerase in 20 μL RNase-free water. .. 2.3 Expression of dsRNA gene using E. coli HT115(DE3) The E. coli strain, HT115(DE3) was obtained from Cold Spring Harbor Laboratory, NY, USA.

    Labeling:

    Article Title: The Zinc-Fingers of KREPA3 Are Essential for the Complete Editing of Mitochondrial mRNAs in Trypanosoma brucei
    Article Snippet: The reaction products were separated by electrophoresis on 10% polyacrylamide gel containing 7M urea and 1X TBE, transferred to Whatman paper, dried, and then visualized after exposure to PhosphorImager screen (GE Healthcare). .. Labeled gRNAs were identified by size in comparison to the labeled low range ssRNA ladder (NEB). ..

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    New England Biolabs ssrna ladder
    <t>mRNA-EGFP</t> transcription. Capped (lane 3) and uncapped (lane 4) mRNAs that code for EGFP were synthesized along with a luciferase mRNA (control, lane 2). Their integrity was visually evaluated using electrophoresis agarose gels stained with GelRed™. Transcripts are compared with the <t>ssRNA</t> ladder (lane 1). Single gel experiment without cropping.
    Ssrna Ladder, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mRNA-EGFP transcription. Capped (lane 3) and uncapped (lane 4) mRNAs that code for EGFP were synthesized along with a luciferase mRNA (control, lane 2). Their integrity was visually evaluated using electrophoresis agarose gels stained with GelRed™. Transcripts are compared with the ssRNA ladder (lane 1). Single gel experiment without cropping.

    Journal: BioMed Research International

    Article Title: VLPs Derived from the CCMV Plant Virus Can Directly Transfect and Deliver Heterologous Genes for Translation into Mammalian Cells

    doi: 10.1155/2019/4630891

    Figure Lengend Snippet: mRNA-EGFP transcription. Capped (lane 3) and uncapped (lane 4) mRNAs that code for EGFP were synthesized along with a luciferase mRNA (control, lane 2). Their integrity was visually evaluated using electrophoresis agarose gels stained with GelRed™. Transcripts are compared with the ssRNA ladder (lane 1). Single gel experiment without cropping.

    Article Snippet: The mRNA was compared with ssRNA ladder (NEB, Ipswich, MA, USA).

    Techniques: Synthesized, Luciferase, Electrophoresis, Staining

    Competition and specificity assays of P9-1. (A) Increasing amounts of unlabeled competitor ssRNA, dsRNA, ssDNA, or dsDNA were mixed with 3.1 nmol DIG-labeled S9-1900nt ssRNA; 4.6 μmol of purified P9-1 was added to each sample, and the sample was

    Journal: Journal of Virology

    Article Title: Viroplasm Protein P9-1 of Rice Black-Streaked Dwarf Virus Preferentially Binds to Single-Stranded RNA in Its Octamer Form, and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site

    doi: 10.1128/JVI.02264-13

    Figure Lengend Snippet: Competition and specificity assays of P9-1. (A) Increasing amounts of unlabeled competitor ssRNA, dsRNA, ssDNA, or dsDNA were mixed with 3.1 nmol DIG-labeled S9-1900nt ssRNA; 4.6 μmol of purified P9-1 was added to each sample, and the sample was

    Article Snippet: The competitors used for competition between ssRNA and dsRNA and between ssDNA and dsDNA were as follows: ssRNA, full-length in vitro -transcribed S9 RNA transcript; dsRNA, annealing product of the plus and minus strands of the full-length S9 RNA; ssDNA, M13mp18 phage (NEB, Beijing, China); dsDNA, PCR amplification product of full-length RBSDV S9.

    Techniques: Labeling, Purification

    Formation of gradually shifted higher-order complexes by P9-1 and RNA. (A) Increasing amounts of P9-1 were incubated with 3.1 nmol of DIG-labeled RBSDV S9-1900nt ssRNA. The complexes were resolved by electrophoresis on a 1% agarose gel, and blots were

    Journal: Journal of Virology

    Article Title: Viroplasm Protein P9-1 of Rice Black-Streaked Dwarf Virus Preferentially Binds to Single-Stranded RNA in Its Octamer Form, and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site

    doi: 10.1128/JVI.02264-13

    Figure Lengend Snippet: Formation of gradually shifted higher-order complexes by P9-1 and RNA. (A) Increasing amounts of P9-1 were incubated with 3.1 nmol of DIG-labeled RBSDV S9-1900nt ssRNA. The complexes were resolved by electrophoresis on a 1% agarose gel, and blots were

    Article Snippet: The competitors used for competition between ssRNA and dsRNA and between ssDNA and dsDNA were as follows: ssRNA, full-length in vitro -transcribed S9 RNA transcript; dsRNA, annealing product of the plus and minus strands of the full-length S9 RNA; ssDNA, M13mp18 phage (NEB, Beijing, China); dsDNA, PCR amplification product of full-length RBSDV S9.

    Techniques: Incubation, Labeling, Electrophoresis, Agarose Gel Electrophoresis

    Predicted RNA-binding domains in MLE. ( A ) Schematic representation of the predicted features of MLE and of the expressed fragments containing individual domains that were analyzed for RNA-binding. All proteins were tagged, either with a C-terminal flag-tag or an N-terminal His 6 -tag (black bars). The numbers represent the MLE aa that delineate the domains. Insert: The purified recombinant MLE derivatives were separated by polyacrylamide gel electrophoresis (PAGE) and stained with Coomassie Blue. Lane M displays a size marker with indicated molecular weights in kDa. ( B ) Scheme of the ssRNA and dsRNA substrates. The sequence of roX2 is depicted in black, Sp6 promoter and T7 promoter are shown in grey. ( C ) Electrophoretic mobility shift assay (EMSA). The indicated amounts of the predicted RNA-binding domains (1–4 pmoles/50–200 nM) were incubated on ice in the presence of 25 fmol (1.25 nM) radiolabeled dsRNA or ssRNA. RNA–protein complexes (bracketed regions) were resolved on a 1.8% agarose gel and visualized by PhosphoImager analysis of the dried gel. Positions of ssRNA and dsRNA are indicated. ( D ) Filter-binding assay. Binding of the predicted RNA-binding domains to dsRNA was analyzed by filter-binding as described in Materials and Methods section. Each sample was analyzed in duplicates and error bars reflect the experimental variation.

    Journal: Nucleic Acids Research

    Article Title: Structure-function analysis of the RNA helicase maleless

    doi: 10.1093/nar/gkm1108

    Figure Lengend Snippet: Predicted RNA-binding domains in MLE. ( A ) Schematic representation of the predicted features of MLE and of the expressed fragments containing individual domains that were analyzed for RNA-binding. All proteins were tagged, either with a C-terminal flag-tag or an N-terminal His 6 -tag (black bars). The numbers represent the MLE aa that delineate the domains. Insert: The purified recombinant MLE derivatives were separated by polyacrylamide gel electrophoresis (PAGE) and stained with Coomassie Blue. Lane M displays a size marker with indicated molecular weights in kDa. ( B ) Scheme of the ssRNA and dsRNA substrates. The sequence of roX2 is depicted in black, Sp6 promoter and T7 promoter are shown in grey. ( C ) Electrophoretic mobility shift assay (EMSA). The indicated amounts of the predicted RNA-binding domains (1–4 pmoles/50–200 nM) were incubated on ice in the presence of 25 fmol (1.25 nM) radiolabeled dsRNA or ssRNA. RNA–protein complexes (bracketed regions) were resolved on a 1.8% agarose gel and visualized by PhosphoImager analysis of the dried gel. Positions of ssRNA and dsRNA are indicated. ( D ) Filter-binding assay. Binding of the predicted RNA-binding domains to dsRNA was analyzed by filter-binding as described in Materials and Methods section. Each sample was analyzed in duplicates and error bars reflect the experimental variation.

    Article Snippet: Electrophoretic mobility shift assay RNA-binding reaction (20 µl) contained radiolabeled ssRNA or dsRNA (25 fmol/1.25 nM) in Band-shift Buffer (20 mM Hepes-KOH pH 7.6, 3 mM MgCl2 , 10% glycerol, 1 mM DTT, 0.1 mg/ml BSA (NEB)).

    Techniques: RNA Binding Assay, FLAG-tag, Purification, Recombinant, Polyacrylamide Gel Electrophoresis, Staining, Marker, Sequencing, Electrophoretic Mobility Shift Assay, Incubation, Agarose Gel Electrophoresis, Filter-binding Assay, Binding Assay

    Stimulation of MLE ATPase activity by RNA. MLE and selected derivatives were analyzed in ATPase assays. 25 fmol (1.25 nM) of the indicated protein and increasing amounts of dsRNA (upper panels) or ssRNA (lower panels) were used in the ATPase reaction as described in Materials and Methods section. In ( B ) different enzyme preparations from ( A ) were used, with slightly different specific activities.

    Journal: Nucleic Acids Research

    Article Title: Structure-function analysis of the RNA helicase maleless

    doi: 10.1093/nar/gkm1108

    Figure Lengend Snippet: Stimulation of MLE ATPase activity by RNA. MLE and selected derivatives were analyzed in ATPase assays. 25 fmol (1.25 nM) of the indicated protein and increasing amounts of dsRNA (upper panels) or ssRNA (lower panels) were used in the ATPase reaction as described in Materials and Methods section. In ( B ) different enzyme preparations from ( A ) were used, with slightly different specific activities.

    Article Snippet: Electrophoretic mobility shift assay RNA-binding reaction (20 µl) contained radiolabeled ssRNA or dsRNA (25 fmol/1.25 nM) in Band-shift Buffer (20 mM Hepes-KOH pH 7.6, 3 mM MgCl2 , 10% glycerol, 1 mM DTT, 0.1 mg/ml BSA (NEB)).

    Techniques: Activity Assay

    Contributions of MLE domains to helicase activity. Helicase reactions were carried out with increasing amounts (1.5, 3, 6, 12 fmole/75, 150, 300, 600 pM) of MLE and derivatives and 25 fmoles (1.25 nM) of radiolabeled dsRNA as described in Materials and Methods section. Lanes 1 and 2 indicate the migration position of the ssRNA (boiled substrate) and dsRNA substrate, respectively. Lanes 3–5 correspond to unwinding reactions from which ATP was omitted. In ( B ) different enzyme preparations from the ones tested in ( A ) were used, with enzyme inputs of 6, 12, 25 fmoles.

    Journal: Nucleic Acids Research

    Article Title: Structure-function analysis of the RNA helicase maleless

    doi: 10.1093/nar/gkm1108

    Figure Lengend Snippet: Contributions of MLE domains to helicase activity. Helicase reactions were carried out with increasing amounts (1.5, 3, 6, 12 fmole/75, 150, 300, 600 pM) of MLE and derivatives and 25 fmoles (1.25 nM) of radiolabeled dsRNA as described in Materials and Methods section. Lanes 1 and 2 indicate the migration position of the ssRNA (boiled substrate) and dsRNA substrate, respectively. Lanes 3–5 correspond to unwinding reactions from which ATP was omitted. In ( B ) different enzyme preparations from the ones tested in ( A ) were used, with enzyme inputs of 6, 12, 25 fmoles.

    Article Snippet: Electrophoretic mobility shift assay RNA-binding reaction (20 µl) contained radiolabeled ssRNA or dsRNA (25 fmol/1.25 nM) in Band-shift Buffer (20 mM Hepes-KOH pH 7.6, 3 mM MgCl2 , 10% glycerol, 1 mM DTT, 0.1 mg/ml BSA (NEB)).

    Techniques: Activity Assay, Migration

    Binding of MLE and MLE derivatives to dsRNA and ssRNA. ( A ) Schematic representation of MLE deletion constructs analyzed in this study as in Figure 1 A. The insert to the right shows the purified recombinant MLE derivatives, separated by PAGE and stained with Coomassie Blue. ( B ) EMSA as in Figure 1 C with protein–RNA complexes (bracketed regions) resolved on a 1.8% agarose gel. Protein amounts range from 6 fmoles (0.3 nM) to 50 fmoles (2.5 nM). ( C ) dsRNA interactions measured by filter-binding as in Figure 1 D.

    Journal: Nucleic Acids Research

    Article Title: Structure-function analysis of the RNA helicase maleless

    doi: 10.1093/nar/gkm1108

    Figure Lengend Snippet: Binding of MLE and MLE derivatives to dsRNA and ssRNA. ( A ) Schematic representation of MLE deletion constructs analyzed in this study as in Figure 1 A. The insert to the right shows the purified recombinant MLE derivatives, separated by PAGE and stained with Coomassie Blue. ( B ) EMSA as in Figure 1 C with protein–RNA complexes (bracketed regions) resolved on a 1.8% agarose gel. Protein amounts range from 6 fmoles (0.3 nM) to 50 fmoles (2.5 nM). ( C ) dsRNA interactions measured by filter-binding as in Figure 1 D.

    Article Snippet: Electrophoretic mobility shift assay RNA-binding reaction (20 µl) contained radiolabeled ssRNA or dsRNA (25 fmol/1.25 nM) in Band-shift Buffer (20 mM Hepes-KOH pH 7.6, 3 mM MgCl2 , 10% glycerol, 1 mM DTT, 0.1 mg/ml BSA (NEB)).

    Techniques: Binding Assay, Construct, Purification, Recombinant, Polyacrylamide Gel Electrophoresis, Staining, Agarose Gel Electrophoresis

    IP RP HPLC analyses of dsRNA and ssRNA under denaturing and non-denaturing conditions. (a) IP RP HPLC chromatogram of dsRNA at 50 °C. (b) IP RP HPLC chromatogram of dsRNA at 75 °C. (c) IP RP HPLC chromatogram of ssRNA at 50 °C. (d) IP RP HPLC chromatogram of ssRNA at 75 °C. (e) IP RP HPLC chromatogram of in vitro transcribed dsRNA with an excess of ssRNA. (f) IP RP HPLC chromatogram of purified dsRNA. Following in vitro transcription, the RNA was purified in a single step using RNase T1/DNase in conjunction with solid phase extraction. 2–3 μg of in vitro transcribed ds/ssRNA was analysed using gradient 1 at 260 nm UV detection.

    Journal: Journal of Chromatography. a

    Article Title: Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry

    doi: 10.1016/j.chroma.2016.12.062

    Figure Lengend Snippet: IP RP HPLC analyses of dsRNA and ssRNA under denaturing and non-denaturing conditions. (a) IP RP HPLC chromatogram of dsRNA at 50 °C. (b) IP RP HPLC chromatogram of dsRNA at 75 °C. (c) IP RP HPLC chromatogram of ssRNA at 50 °C. (d) IP RP HPLC chromatogram of ssRNA at 75 °C. (e) IP RP HPLC chromatogram of in vitro transcribed dsRNA with an excess of ssRNA. (f) IP RP HPLC chromatogram of purified dsRNA. Following in vitro transcription, the RNA was purified in a single step using RNase T1/DNase in conjunction with solid phase extraction. 2–3 μg of in vitro transcribed ds/ssRNA was analysed using gradient 1 at 260 nm UV detection.

    Article Snippet: The following PCR parameters were used: the initial denaturation was 1 cycle of 30 s at 98 °C, 30 cycles of 30 s at 98 °C, 30 s at 68 °C, and 30 s at 72 °C and a final extension at 72 °C for 2 mins. dsRNA and ssRNA were also generated using in vitro transcription in conjunction with HiScribe™ T7 High Yield RNA Synthesis Kit (New England Biolabs): 10 mM NTPs, 1 x reaction buffer, 1 μg DNA template and 2 μL HiScribe™ T7 polymerase in 20 μL RNase-free water.

    Techniques: High Performance Liquid Chromatography, In Vitro, Purification

    Purification of dsRNA from E. coli . (a) Agarose gel electrophoresis of purified in vitro transcribed dsRNA and ssRNA. Following in vitro transcription, 2 μg of dsRNA containing excess ssRNA were incubated with RNase T1 in both the presence and absence of 0.5 M NaCl and purified by SPE. (b) Agarose gel electrophoresis of extracted and purified dsRNA. Following TRIzol extraction of total RNA from E. coli expressing dsRNA samples were incubated with RNase T1 in both the presence and absence of 0.3 M NaCl as indicated prior to purification using solid phase extraction. Control experiments were performed using total RNA from non-induced E. coli . (c) IP RP HPLC chromatogram of purified dsRNA. Following TRIzol extraction the total RNA from E. coli was purified in a single step using RNase T1/DNase in conjunction with solid phase extraction prior to analysis using IP RP HPLC gradient condition 1 at 260 nm UV detection.

    Journal: Journal of Chromatography. a

    Article Title: Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry

    doi: 10.1016/j.chroma.2016.12.062

    Figure Lengend Snippet: Purification of dsRNA from E. coli . (a) Agarose gel electrophoresis of purified in vitro transcribed dsRNA and ssRNA. Following in vitro transcription, 2 μg of dsRNA containing excess ssRNA were incubated with RNase T1 in both the presence and absence of 0.5 M NaCl and purified by SPE. (b) Agarose gel electrophoresis of extracted and purified dsRNA. Following TRIzol extraction of total RNA from E. coli expressing dsRNA samples were incubated with RNase T1 in both the presence and absence of 0.3 M NaCl as indicated prior to purification using solid phase extraction. Control experiments were performed using total RNA from non-induced E. coli . (c) IP RP HPLC chromatogram of purified dsRNA. Following TRIzol extraction the total RNA from E. coli was purified in a single step using RNase T1/DNase in conjunction with solid phase extraction prior to analysis using IP RP HPLC gradient condition 1 at 260 nm UV detection.

    Article Snippet: The following PCR parameters were used: the initial denaturation was 1 cycle of 30 s at 98 °C, 30 cycles of 30 s at 98 °C, 30 s at 68 °C, and 30 s at 72 °C and a final extension at 72 °C for 2 mins. dsRNA and ssRNA were also generated using in vitro transcription in conjunction with HiScribe™ T7 High Yield RNA Synthesis Kit (New England Biolabs): 10 mM NTPs, 1 x reaction buffer, 1 μg DNA template and 2 μL HiScribe™ T7 polymerase in 20 μL RNase-free water.

    Techniques: Purification, Agarose Gel Electrophoresis, In Vitro, Incubation, Expressing, High Performance Liquid Chromatography