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| 货号 (SKU) | 包装规格 | 是否现货 | 价格 | 数量 |
|---|---|---|---|---|
| R749972-200U |
200U |
期货  |
| |
| R749972-1KU |
1KU |
期货 |
| |
| R749972-5KU |
5KU |
期货 |
|
| 规格或纯度 | 不含除RNase III之外的其它种类的RNA内切酶和外切酶，不含DNase。 |
|---|---|
| 稳定性与储存 | -20ºC保存，两年有效。 |
| 英文名称 | RNase III (dsRNA-specific) |
| 储存温度 | -20°C储存 |
| 运输条件 | 超低温冰袋运输 |
| 产品介绍 |
产品介绍： 阿拉丁生产的RNase III，即核糖核酸酶III，是由阿拉丁自主研发的PerfectProtein™技术平台表达、纯化获得的一种来源于大肠杆菌的双链RNA (Double-strand RNA, dsRNA)特异性的核糖核酸酶。在锰离子(Mn2+', ")存在的反应缓冲液中，RNase III可特异性地将较长的双链RNA (dsRNA)切割成长度约为18-25bp的3'羟基末端带有2-3个突出碱基的干扰RNA (siRNA) 1,2]，该产物与Dicer酶切产生的底物类似，具有用于哺乳动物细胞的RNA干扰(RNA interference)、基因沉默、靶标确认等多种用途3]。RNase III不能水解DNA或单链RNA。阿拉丁生产的RNase III用于切割500bp双链RNA生成siRNA的效果请参考图1。图1. 阿拉丁生产的RNase III 切割500bp双链RNA生成siRNA的效果图。使用本产品或N公司(Competitor)的RNase III，在10µl反应体系(50mM Tris-HCl, 50mM NaCl, 1mM DTT, 20µM MnCl2 (pH7.5 @25ºC))中加入1µg 500bp的dsRNA，以及图中指定量的本产品或N公司(Competitor)的RNase III，然后用超纯水补至10µl，37ºC孵育20分钟进行反应。反应完成后立即加入终浓度为50mM的EDTA以终止反应。取反应后产物10μl加入2μl 6X DNA Loading Buffer ，1%的琼脂糖凝胶电泳，随后在紫外灯下观察实验结果。如图所示，本产品与N公司的RNase III产品相比，对500bp的dsRNA具有类似的酶切效果。dsRNA是使用T7 Quick High Yield RNA Transcription Kit 将两条含T7启动子的500bp的互补DNA链进行体外转录生成两条互补的500nt单链RNA，随后使用Annealing Buffer for RNA Oligos (5X) 并按照该产品说明书推荐的程序，把这两条互补的500nt单链RNA进行退火反应得到的产物。实际操作时不同实验条件获得的实验结果会略有差异，图中所示结果仅供参考。用途： dsRNA的水解酶切；产生siRNA；基因沉默；基因靶标确认 (target validation)。来源： 纯化自携带编码大肠杆菌RNase III基因的E.coli重组菌株。酶储存溶液： 10mM Tris-HCl, 500mM NaCl, 1mM DTT, 0.5mM EDTA, 50% Glycerol (pH8.0 @25ºC)。10X Reaction Buffer: 500mM Tris-HCl, 500mM NaCl, 10mM DTT (pH7.5 @25ºC).失活或抑制： 不能进行热失活；加入反应终止液10X EDTA(终浓度为50mM EDTA)可使RNase III失活。注意事项： RNase III不能进行热失活，否则会降低siRNA的产率；可通过加入终浓度为50mM的EDTA以终止反应。RNA极易降解，在整个操作过程中应尽可能避免环境中RNase的影响。RNase III使用时宜存放在冰盒内或冰浴上，使用完毕后宜立即放置于-20℃保存。本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。为了您的安全和健康，请穿实验服并戴一次性手套操作。使用说明： 1.siRNA的制备：a.对于siRNA的制备，参考下表在冰浴中配制如下反应体系(以20和100μl体系为例)：ReagentVolumeVolumeFinal ConcentrationdsRNAxμl (2μg)xμl (10μg)100ng/µl10X Reaction Buffer2μl10μl1X10X MnCl22μl10μl1XNuclease-Free Water(14-x)μl(70-x)μl-RNase III2μl10μl0.2U/µlTotal Volume20μl100μl注1：为获得最好的底物酶切效果，实验过程中可参考酶活单位定义对酶用量进行适当调整，摸索最优反应体系。注2：如果同时进行多个反应，可以把上表中除RNase III之外所有成分提前混合，分装到各反应管中，最后再加入RNase III。b.按上表设置好反应体系后，适当轻轻混匀反应体系，随后低速离心以使粘附在管壁上的液体沉淀至管底。c.反应条件：37ºC孵育20分钟。注：反应时间可以根据实际情况酌情适当调节。d.终止反应：加入反应终止液10X EDTA(终浓度为50mM EDTA)使RNase III失去活性。注：不能进行热失活，热失活会降低siRNA的产率。2.其它用途可以参考适当的文献资料进行。参考文献：1.Morlighem JE, Petit C, Tzertzinis G. Biotechniques. 2007. 42(5):599-600, 602, 604-6.2.Court DL, Gan J, Liang YH, Shaw GX, Tropea JE, et al. Annu Rev Genet. 2013. 47:405-31.3.Donzé O, Picard D. Nucleic Acids Res. 2002. 30(10):e46.Aladdin's RNase III is a dsDNA-specific ribonuclease derived from Escherichia coli, expressed and purified using the PerfectProtein™ Technology Platform developed by aladdin. This product specifically cleaves double-stranded RNA (dsRNA) in the presence of manganese (Mn2+), producing 18-25bp interference RNA (siRNA) with 2-3 protruding bases at the 3' hydroxyl end [1,2]. Similar to the products generated by Dicer enzyme, the siRNA has various applications such as RNA interference, gene silencing, and target validation in mammalian cells [3]. RNase III cannot hydrolyze DNA or single-stranded RNA.Please refer to Figure 1 for the cleavage of dsRNA (500bp) by this product to generate siRNA.Figure 1. Generation of siRNA from dsRNA (500bp) using Aladdin's RNase III . In a 10µl reaction (50mM Tris-HCl, 50mM NaCl, 1mM DTT, 20µM MnCl2 (pH7.5 at 25℃)), add 1µg of dsRNA (500bp) and a specified amount of this product or N Company's (Competitor) RNase III as indicated in the figure. The reaction was incubated at 37℃ for 20 minutes, then terminated by adding EDTA. The reaction product was mixed with 6X DNA Loading Buffer , and subjected to 1% agarose gel electrophoresis. The experimental results were observed under UV light. As shown in the figure, this product exhibited similar performance to N Company's RNase III. The dsRNA was generated by in vitro transcription of dsDNA containing a T7 promoter using the T7 Quick High Yield Transcription Kit . The resulting complementary 500nt single-stranded RNAs were annealed using the Annealing Buffer for RNA Oligos (5X) following the product manual. This figure is for reference only, which may vary depending on different experimental conditions.s Application： Hydrolysis of dsRNA; production of siRNA; gene silencing; target validation.Source： Purified from E. coli with expression of recombinant RNase III.Enzyme storage buffer： 10mM Tris-HCl, 500mM NaCl, 1mM DTT, 0.5mM EDTA, 50% Glycerol (pH8.0 at 25℃).Inactivation or inhibition： This enzyme can be inactivated by EDTA at a final concentration of 50mM. Heat inactivation of this enzyme can not be performed.Precautions： The reaction can be terminated by adding EDTA to a final concentration of 50mM. Do not perform heat inactivation of RNase III. Otherwise, the yield of siRNA will be reduced.RNA is highly susceptible to degradation. Please handle RNA samples with extreme care to avoid RNase contamination.RNase III should be kept on ice during use, and should be stored at -20°C immediately after use.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. Preparation of siRNAa. Set up the reaction (total volume of 20 or 100μl) on ice as follows:ReagentVolumeVolumeFinal ConcentrationdsRNAxμl (2μg)xμl (10μg)100ng/µl10X Reaction Buffer2μl10μl1X10X MnCl22μl10μl1XNuclease-Free Water(14-x)μl(70-x)μl-RNase III2μl10μl0.2U/µlTotal Volume20μl100μl-Note 1: To achieve the best digestion effect, the enzyme dosage can be adjusted as appropriate. Note 2: If multiple reactions are carried out simultaneously, prepare a mastermix of all components in the table except for RNase III. Aliquot the mastermix into each reaction tube, then add RNase III at the end. b. Mix the reaction well gently and centrifuge briefly to collect liquid to the bottom. c. Incubate at 37℃ for 20 minutes. Note: The reaction time can be adjusted appropriately as required. d. Terminate the reaction by adding 10X EDTA to inactivate RNase III. Note: Heat inactivation should not be performed as it reduces the yield of siRNA.2. For other applications, please refer to relevant literature.References:1. Morlighem JE, Petit C, Tzertzinis G. Biotechniques. 2007. 42(5):599-600, 602, 604-6.2. Court DL, Gan J, Liang YH, Shaw GX, Tropea JE, et al. Annu Rev Genet. 2013. 47:405-31.3. Donzé O, Picard D. Nucleic Acids Res. 2002. 30(10):e46. |
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