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E coli rnase h

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E. coli RNase H is an enzyme that specifically degrades the RNA strand in RNA-DNA hybrids. It is isolated from the bacterium Escherichia coli and is commonly used in molecular biology applications.

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Lab products found in correlation

Formamide, by Fujifilm (2 mentions) 0.5 m edta, by Nippon Gene (2 mentions) Novex tbe urea sample buffer, by Thermo Fisher Scientific (2 mentions) Microrna marker, by New England Biolabs (2 mentions) Mini protean 15 tbe urea gel, by Bio-Rad (2 mentions) Sybr green 2, by Thermo Fisher Scientific (2 mentions) Gel doctm ez system, by Bio-Rad (2 mentions) Rnase h, by New England Biolabs (2 mentions) Recombinant human rnase h1, by RayBiotech (2 mentions) Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Quant it rna assay kit, by Thermo Fisher Scientific (1 mentions) E coli dna ligase, by New England Biolabs (1 mentions) Superscript 3 first strand synthesis system for rt pcr kit, by Thermo Fisher Scientific (1 mentions) E coli dna polymerase, by New England Biolabs (1 mentions) Agencourt rnaclean xp spri beads, by Beckman Coulter (1 mentions) E coli dna polymerase 1, by New England Biolabs (1 mentions) Second strand synthesis reaction buffer, by New England Biolabs (1 mentions) E coli ligase, by New England Biolabs (1 mentions) Dnase 1, by Promega (1 mentions)

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RNase H (344 citations)

6 protocols using e coli rnase h

1

RNase H Cleavage Assays

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E.coli RNase H assays were performed by incubating co-annealed 1 uM substrate (either Sub_KRas12 [wt] or [G12D], or Sub_KRas12_long [G12D]) and 5 uM active XNAzyme FR6_1_KRas12B, inactive FR6_1_KRas12Bi, or DNAzyme 10-23_KRasC ([6+7] and [10+10] versions) with 0.05 unit/uL E.coli RNase H (NEB, USA) in NEB RNase H buffer (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 10 mM DTT, 3 mM MgCl2), according to the manufacturer’s instructions (20 min at 37 °C). Human RNase H assays were performed using co-annealed 0.25 uM substrate and 0.25 uM enzyme, 0.01 ug/ul recombinant human RNase H1 (RayBiotech, USA) in 20 mM Tris-HCl (pH 7.5), 20 mM KCl, 5 mM DTT, 1 mM MgCl2 (1 h at 37 °C). Reactions were stopped by the addition of an excess of PAGE loading buffer and snap-frozen on dry ice.
Taylor A.I., Wan C.J., Donde M.J., Peak-Chew S.Y, & Holliger P. (2022). A modular XNAzyme cleaves long, structured RNAs under physiological conditions and enables allele-specific gene silencing. Nature chemistry, 14(11), 1295-1305.
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2

RNase H Cleavage Assays

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E.coli RNase H assays were performed by incubating co-annealed 1 uM substrate (either Sub_KRas12 [wt] or [G12D], or Sub_KRas12_long [G12D]) and 5 uM active XNAzyme FR6_1_KRas12B, inactive FR6_1_KRas12Bi, or DNAzyme 10-23_KRasC ([6+7] and [10+10] versions) with 0.05 unit/uL E.coli RNase H (NEB, USA) in NEB RNase H buffer (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 10 mM DTT, 3 mM MgCl2), according to the manufacturer’s instructions (20 min at 37 °C). Human RNase H assays were performed using co-annealed 0.25 uM substrate and 0.25 uM enzyme, 0.01 ug/ul recombinant human RNase H1 (RayBiotech, USA) in 20 mM Tris-HCl (pH 7.5), 20 mM KCl, 5 mM DTT, 1 mM MgCl2 (1 h at 37 °C). Reactions were stopped by the addition of an excess of PAGE loading buffer and snap-frozen on dry ice.
Taylor A.I., Wan C.J., Donde M.J., Peak-Chew S.Y, & Holliger P. (2022). A modular XNAzyme cleaves long, structured RNAs under physiological conditions and enables allele-specific gene silencing. Nature chemistry, 14(11), 1295-1305.
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3

Kinetics of LNA Gapmer-Mediated JEV RNA Cleavage

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A target 17-mer JEV RNA sequence complementary to 3′ UTR stem-loop-targeted LNA gapmers was synthesized with HPLC grade by Ajinomoto Bio-Pharma Services GeneDesign, Inc. LNA gapmers (1.5 µM), synthesized JEV RNA (6 µM), and E. coli RNase H (50 units/mL; New England Biolabs, Ipswich, MA, USA) were mixed and incubated in 1 × RNase H reaction buffer at 37 °C for 120 min. The 20 µL reaction mixture was collected at 0, 5, 10, 30, 60, and 120 min after the reaction. The collected reaction mixture was immediately quenched by adding 1 µL of 0.5 M EDTA (NIPPON GENE, Tokyo, Japan) at pH 8.0 and denatured with an equal amount of 21 µL formamide (Wako). The samples were immediately used for a subsequent procedure or stored at −80 °C until use. They were mixed with an equal volume of NovexTM TBE-Urea sample buffer (Thermo Fisher Scientific), heated at 70 °C for 4 min, and immediately placed on ice. Aliquots having a volume of 20 µL and 12 µL microRNA marker (New England Biolabs) were submitted to a Mini-PROTEAN 15% TBE-urea gel with ten wells (Bio-Rad, Santa Rosa, CA, USA) and electrophoresed at 200 V for 30 min. The gel was stained by SYBR® Green II (Thermo Fisher Scientific), and images were captured using the Gel DocTM EZ system (Bio-Rad). The band intensities were quantified using ImageJ software (version 1.53k; NIH).
Okamoto S., Echigoya Y., Tago A., Segawa T., Sato Y, & Itou T. (2023). Antiviral Efficacy of RNase H-Dependent Gapmer Antisense Oligonucleotides against Japanese Encephalitis Virus. International Journal of Molecular Sciences, 24(19), 14846.
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4

Kinetics of LNA Gapmer-Mediated JEV RNA Cleavage

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A target 17-mer JEV RNA sequence complementary to 3′ UTR stem-loop-targeted LNA gapmers was synthesized with HPLC grade by Ajinomoto Bio-Pharma Services GeneDesign, Inc. LNA gapmers (1.5 µM), synthesized JEV RNA (6 µM), and E. coli RNase H (50 units/mL; New England Biolabs, Ipswich, MA, USA) were mixed and incubated in 1 × RNase H reaction buffer at 37 °C for 120 min. The 20 µL reaction mixture was collected at 0, 5, 10, 30, 60, and 120 min after the reaction. The collected reaction mixture was immediately quenched by adding 1 µL of 0.5 M EDTA (NIPPON GENE, Tokyo, Japan) at pH 8.0 and denatured with an equal amount of 21 µL formamide (Wako). The samples were immediately used for a subsequent procedure or stored at −80 °C until use. They were mixed with an equal volume of NovexTM TBE-Urea sample buffer (Thermo Fisher Scientific), heated at 70 °C for 4 min, and immediately placed on ice. Aliquots having a volume of 20 µL and 12 µL microRNA marker (New England Biolabs) were submitted to a Mini-PROTEAN 15% TBE-urea gel with ten wells (Bio-Rad, Santa Rosa, CA, USA) and electrophoresed at 200 V for 30 min. The gel was stained by SYBR® Green II (Thermo Fisher Scientific), and images were captured using the Gel DocTM EZ system (Bio-Rad). The band intensities were quantified using ImageJ software (version 1.53k; NIH).
Okamoto S., Echigoya Y., Tago A., Segawa T., Sato Y, & Itou T. (2023). Antiviral Efficacy of RNase H-Dependent Gapmer Antisense Oligonucleotides against Japanese Encephalitis Virus. International Journal of Molecular Sciences, 24(19), 14846.
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5

RNA Purification and cDNA Synthesis

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Briefly, 30 µL of vRNAs or 30 µL of IVT RNA mixes were purified and concentrated to 10 µL using Agencourt RNAClean XP SPRI beads (Beckman Coulter) and subjected to first strand cDNA synthesis using the Superscript III First-Strand Synthesis System for RT-PCR Kit (Thermo Fisher Scientific) and a mixture of random hexamers and U12–U13 specific primers (see above). The second cDNA strand was synthetized using 5 U of E. coli RNase H, 40 U of E. coli DNA polymerase, and 10 U of E. coli DNA ligase for 2 h at 16°C (New England Biolabs). Double-stranded cDNAs were purified using Agencourt RNAClean XP SPRI beads (Beckman Coulter) and quantified using the Quant-iT RNA Assay Kit (Thermo Fisher Scientific).
Boussier J., Munier S., Achouri E., Meyer B., Crescenzo-Chaigne B., Behillil S., Enouf V., Vignuzzi M., van der Werf S, & Naffakh N. (2020). RNA-seq accuracy and reproducibility for the mapping and quantification of influenza defective viral genomes. RNA, 26(12), 1905-1918.
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6

Total RNA Extraction and cDNA Synthesis

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Total RNA was extracted from ca. 100 mg of floral buds or leaves, with Trizol Reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA) following manufacturer's instructions, and re-purified using either RNeasy Plant Mini Kit (QIAGEN) or EasyPure Plant RNA kit (Transgen Biotech, Beijing, China) . After verification of RNA integrity by agarose gel electrophoresis (1 % w/v), RNA was treated with 4 U of DNase I (Promega, Madison, Wisconsin, USA). Simple-stranded cDNA was synthesized using the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific) and oligo dT 20 as the primer, following the manufacturer's protocol. Then, double-stranded cDNA was obtained in a 150 µl-reaction containing 1X Second Strand Synthesis Reaction Buffer (New England Biolabs, NEB, Hitchin, Hertfordshire, UK), 2 mM dNTP mix, 10 U of E. coli ligase (NEB), 40 U of E. coli DNA polymerase I (NEB), 2 U of E. coli RNase H (NEB) and DEPC-treated water. The mix was incubated for 2 h at 16 ºC. Double-stranded cDNA was purified with one volume of phenol:chloroform:isoamyl alcohol (25:24:1), precipitated with NH 4 OAc 7.5 M and resuspended in 10 mM Tris-HCl.
Cascales J., Acevedo R.M., Paiva D.I, & Gottlieb A.M. (2021). Differential DNA methylation and gene expression during development of reproductive and vegetative organs in Ilex species. Journal of plant research, 134(3).
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