epicentre*** Q***1250 Q-Beta Replicase

Q-Beta***酶（QβReplicase）是一种RNA定向的RNA聚合酶，负责***噬菌体Q-Beta RNA***组。Blumenthal和Carmichael在一篇评论中介绍了酶的各个方面。1它由四个亚基组成，其中一个由Q-β噬菌体编码，三个由大肠***宿主编码。EPICENTRE的Q-Beta***酶是从大肠***中纯化的，其中含有表达噬菌体编码亚基的质粒。所有四个酶亚基均等比例存在（图1）。

Q-Beta Replicase (Qβ Replicase) is an RNA-directed RNA polymerase that is resp***ible for replication of the coliphage Q-Beta RNA genome. Various aspects of the enzyme are presented in a review by Blumenthal and Carmichael.1 It is composed of four subunits, one of which is encoded by the Q-Beta phage and three by the E. coli host. EPICENTRE's Q-Beta Replicase is purified from E. coli containing a pla***id that expresses the phage-encoded subunit. All four enzyme subunits are present in equal proporti*** (Fig. 1).

在体外，Q-Beta***酶可以利用Q-Beta噬菌体RNA以外的其他RNA分子作为模板。这些包括在Q-β***酶反应中发现的各种亚***组变异RNA分子，例如中变（MDV）RNA和其他较小的变异RNA。另外，它也可以使用其他“非天然”模板，例如聚（rC），带有寡核糖核苷酸的RNA或带有多个C残基的RNA。Q-Beta***酶也可以***由包埋在中等变数MDV-1 RNA中的RNA序列组成的重组RNA分子。2该酶使用MDV-1 RNA或重组MDV-1 RNA的两条链作为模板，只要酶过量，产物的合成就呈指数级。在等温条件下，可以在短时间内实现重组序列的大扩增。

In vitro, Q-Beta Replicase can utilize other RNA molecules besides the Q-Beta phage RNA as templates. These include various subgenomic variant RNA molecules that were found in Q-Beta Replicase reacti***, such as midivariant (MDV) RNAs, and other ***aller variant RNAs. In addition, it can also use other "non-natural" templates such as poly(rC), RNA primed with an oligoribonucleotide, or RNA tailed with several C residues. Recombinant RNA molecules c***isting of RNA sequences embedded within midivariant MDV-1 RNA can also be replicated by Q-Beta Replicase.2 The enzyme uses both strands of MDV-1 RNA or recombinant MDV-1 RNAs as templates, and synthesis of product is exponential so long as the enzyme is in excess. Large amplification of recombinant sequences can be achieved in a short time under isothermal conditi***.

单位定义：一个Q-Beta***酶单位在30°C下使用20 ng / µl的poly（rC）作为模板，在包含33 mM的反应混合物中，在30°C的10分钟内催化将1 nmole rGTP掺入poly（rG）中。三***酯（pH 7.5），66 mM***钾，10 mM***镁，0.5 mM DTT和1 mM rGTP。

Unit Definition: One unit of Q-Beta Replicase catalyzes the incorporation of 1 nmole of rGTP into poly(rG) in 10 minutes at 30°C using 20 ng/µl of poly(rC) as a template in a reaction mixture containing 33 mM Tris-acetate (pH 7.5), 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM DTT, and 1 mM rGTP.

储存缓冲液和条件：含有50 mM Tris-HCl（pH 7.5），100 mM N***，1 mM DTT，0.1 mM EDTA和0.1％Triton X-100的50％甘油。储存在-20°C的冰箱中，无霜循环。

Storage Buffer & Conditi***: 50% glycerol containing 50 mM Tris-HCl (pH 7.5), 100 mM N***, 1 mM DTT, 0.1 mM EDTA, and 0.1% Triton X-100. Store at -20°C in a freezer without a defrost cycle.

10X TA反应缓冲液（另售）： 330 mM三***酯（pH 7.5），660 mM***钾，100 mM***镁和5 mM DTT。

Quality Control: The physical purity of Q-Beta Replicase is greater than 95%, as determined by SDS polyacrylamide gel electrophoresis. All four subunits are present in equal amounts. In order to minimize the possibility of contamination of the Q-Beta Replicase by Q-Beta templates, EPICENTRE uses only poly(rC) as a template for determining activity. Enzymatic assays of Q-Beta Replicase activity without added template produce no detectable RNA by gel analysis, thus ruling out contamination with RNA that is exponentially amplifiable by the enzyme. Gel analysis also indicates the absence of contamination by E. coli DNA-dependent RNA polymerase. The enzyme is free of detectable contaminating exo- and endonuclease and RNase activities.

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