≥95%, free of DNA endonuclease, exonuclease and other ribonuclease.
Stability And Storage
Store at -20℃, valid for at least 1 year.
Storage Temp
Store at -20°C
Shipped In
Ice chest + Ice pads
Product Description
Aladdin's Thermostable RNase H is an endoribonuclease that remains active at high temperature and specifically hydrolyzes RNA in DNA-RNA hybrids. It cannot hydrolyze the phosphodiester bonds of single- or double-stranded RNA or DNA.Thermostable RNase H remains high enzyme activity at temperature above 65℃. Its half-life is approximately several hours at 70℃ and approximately 30min at 95℃. Its high-temperature tolerance enables more specific degradation of the RNA strand in RNA-DNA hybrids at high temperatures. Thermostable RNase H and E. coli RNase H have similar enzymatic functions, but E. coli RNase H will be inactivated at above 55℃.Please refer to Figure 1 for the comparison of thermal stability of Thermostable RNase H and E. coli RNase H.Figure 1. The comparison of thermal stability of Aladdin's Thermostable RNase H and E. coli RNase H. In 20µl reactions, different amounts of Thermosable RNase H or E. coli RNase H were added as indicated in the figure, and incubated at 65℃ for 20 minutes, after which RNA-DNA hybrids were added to perform the digestion with Thermostable RNase H at 50℃ for 20 minutes or E. coli RNase H at 37℃ for 20 minutes. After incubation, the reactions were immediately transfer into ice for 3-5 minutes and 1μl of 0.5M EDTA was added to each each tube to terminate the reactions. The reaction products were resolved on a 15% non-denaturing polyacrylamide gel and image was taken after nucleic acid staining with NA-Red . As shown in the figure, E. coli RNase H loses activity at 65℃, while Thermostable RNase H remains active. Reaction mix (20μl) for Thermostable RNase H
Source：
Recombinant protein expressed in E. coli.Definition of enzyme activity unit: One unit is defined as the amount of enzyme required to produce 1 nmol of ribonucleotides from 40pmol of a fluorescently labeled 25 base pair RNA-DNA hybrid in a total reaction volume of 50µl in 20 minutes at 50℃.Purity: ＞95%, free of DNA endonuclease, exonuclease and other ribonuclease.
This product can be degraded by protease K or inhibited by 25mM EDTA.
Precautions：
The optimal reaction temperature of this product is higher than 65℃. Its activity at 65℃ is 3-4 times of that at 37℃. It remains active at 95℃.This protocol uses the reaction temperature of 50℃. In practice, the reaction temperature can be increased appropriately to enhance its activity.The reaction buffer of this product contains MgCl2. When Thermomostable RNase H is applied to RNA-DNA hybrid or RNA samples at high temperatures, the reaction time and temperature should be controlled appropriately to avoid the metal-mediated degradation of ssRNA .This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use：
1.Annealing of RNA-DNA hybrids.Mix ssRNA and ssDNA in equal molar to the recommended final concentration of 20µM. Incubate at 90℃ for 1min, and then cool to 25℃ gradiently to form RNA-DNA hybrids. The annealing can be performed in ’s Annealing Buffer for RNA Oligos according to the instruction manual of the product. The annealing products can be stored at -80℃ for future use.2.For the degradation of the RNA strand in RNA-DNA hybrids, set up the following reaction in a nuclease-free microfuge tube on ice:ReagentVolumeFinal ConcentrationDEPC-treated Water(17-x)µl-RNA-DNA hybridsxµl0.1µg/µl or less10X RNase H Reaction Buffer2µl1XThermostable RNase H (5U/µl)1µl0.25U/µlTotal Volume20µl-Note:a.Avoid RNase contamination. Treat reagents and consumables required with DEPC to remove RNase.b.When multiple reactions are required, prepare a master mix including all reagents except for RNA-DNA hybrids and then dispense to different nuclease-free tubes .c.The amount of RNA-DNA hybrids should not exceed 2μg in the total reaction volume of 20µl.3.Incubate at 50℃ for 20min.If the digestion effect is not good, increase the reaction temperature to 65℃ or higher based on the stability of the RNA-DNA hybrids (the enzyme activity at 65℃ is about 3 times that at 50℃), or consider extending the reaction time appropriately. To remove specific RNA from total RNA, the reaction temperature should be optimized by users.4.Add proteinase K or 1µl of 0.5M EDTA (pH8.0) to stop the reaction.Related Products:Cat. No.Product NamePack SizeD7089RNase H100UR7090SThermostable RNase H250UR7090MThermostable RNase H1000UR7090LThermostable RNase H5000U
