Uracil-DNA Glycosylase (Heat-labile, Cod)

阿拉丁生产的Uracil-DNA Glycosylase (Heat-labile, Cod)，也称Heat-labile Cod UDG，即热敏型鳕鱼UDG，是来源于大西洋鳕鱼(Gadus morhua cod)，可催化含尿嘧啶的DNA链中的尿嘧啶(dU)碱基和脱氧核糖之间的N-糖苷键发生水解，从而释放游离尿嘧啶。Uracil-DNA Glycosylase (UDG)可以水解含有dU的单链或双链DNA，但不能水解RNA或含有dU的长度不超过6个碱基DNA寡聚体。UDG主要应用于消除PCR扩增过程中带来的产物污染问题。其防止污染的原理为：在PCR反应中加入适量的dUTP，以dUTP替代dTTP掺入DNA中，形成含dU碱基的PCR扩增产物；后续进行PCR反应时，使用UDG酶选择性切割可能被污染而带入的之前PCR扩增产生的含有dU的单链或双链DNA，从而避免之前的PCR扩增产物可能的污染对于本次PCR扩增带来的负面影响。本产品为热敏型，在50℃孵育10分钟可以迅速不可逆灭活。在PCR或RT-PCR反应前，PCR或RT-PCR体系中加入Heat-labile Cod UDG室温处理5min，即可充分消除可能的之前的含有dUTP的PCR扩增产物的污染。本产品不仅适用于PCR，也适用于qPCR、RT-PCR和qRT-PCR等体系。活性定义：One unit is the amount of enzyme required to liberate 1 nmol uracil from dU-containing DNA in one hour at 37℃.阿拉丁生产的Heat-labile Cod UDG酶活性鉴定结果可参考图1。图1.阿拉丁和N公司的Heat-labile Cod UDG催化酶解5µl含dU碱基的PCR扩增产物效果图。使用本产品或国外N公司的酶，在20µl体系，分别以5µl含dU碱基的PCR扩增1500bp PCR产物为底物和不同量的(0U, 0.01U, 0.02U, 0.04U, 0.2U) Heat-labile Cod UDG，在1X Heat-labile Cod UDG Buffer，37℃孵育1h，然后进行1%的琼脂糖凝胶电泳检测。如图所示，本产品与N公司相比，具有相当的酶活。M, DNA marker )。阿拉丁生产的 Heat-labile Cod UDG 经灭活处理后酶活鉴定结果可参考图 2。图2.阿拉丁和N公司的Heat-labile Cod UDG以及阿拉丁生产的E. coli UDG 经50℃10min处理后，催化酶解5µl含dU碱基的PCR扩增产物效果图。将不同量的E. coli UDG (0U, 0.005U, 0.01U, 0.025U)和阿拉丁以及N公司不同量的Heat-labile Cod UDG (0U, 0.02U, 0.04U, 0.2U)经50℃ 10min处理之后，在20µl体系，分别以5µl含dU碱基的PCR扩增1500bp PCR产物为底物，在1X Heat-labile Cod UDG Buffer，37℃孵育1h，然后进行1%的琼脂糖凝胶电泳检测。如图所示，E. coli UDG50℃处理10min之后酶活仅轻微下降，而阿拉丁和N公司的Heat-labile Cod UDG均完全灭活。M, DNA marker )。 用途 ： 去除含尿嘧啶的单链或双链DNA；去除含dU的PCR产物气溶胶污染；DNA修复与突变检测；提高PCR产物的克隆效率；提高点诱变的效率；研究蛋白质-DNA相互作用。用于PCR、qPCR、RT-PCR、RT-qPCR、one-step RT-PCR体系。 来源 ： 大肠杆菌重组、表达和纯化而获得。 酶储存溶液 ： 50mM Tris-HCl (pH 7.5), 100mM NaCl, 0.5mM EDTA, 1mM DTT,0.1% Triton X-100, 50% (v/v) glycerol。10X Heat-labile Cod UDG Buffer：700mM Tris-HCl, 100mM NaCl, 10mM EDTA, 1mg/ml BSA, pH 8.0 at 25℃。 失活或抑制 ： 50℃加热10min可以使Heat-labile Cod UDG完全失活。 注意事项 ： Heat-labile Cod UDG酶在大多数PCR反应缓冲液中均具有活性，但对于自行使用的PCR或RT-PCR体系，首次使用时建议先测试一下是否和所使用的体系兼容。通常取含dUTP的PCR扩增产物，参考图1加入适量UDG，观察能否有效降解含dUTP的PCR扩增产物。dNTP/dUTP推荐选购阿拉丁的D7376 dNTP/dUTP Mixture (2.5mM each/5mM)。Heat-labile Cod UDG长期储存请置于-80℃保存，常规使用请于-20℃保存。-80℃保存会结冻，尽量避免反复冻融。如果需要将Heat-labile Cod UDG酶应用于One-Step RT-PCR或者One-Step qRT-PCR体系，需要选择耐热反转录酶，并需要将逆转录温度设置为50℃-55℃。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.按照常规反应体系设置PCR或RT-PCR反应体系，同时加入Heat-labile Cod UDG至最终浓度为0.01U/μl，混匀。通常仅加入PCR或RT-PCR的buffer即可，无需加入UDG的buffer。2.25℃孵育5min (去除可能的含dUTP的PCR扩增产物的污染)，随后进入PCR或RT-PCR程序。 Aladdin's Uracil-DNA Glycosylase (Heat-labile, Cod), also known as Heat-labile Cod UDG, is a thermolabile UDG derived from Atlantic cod (Gadus morhua) and catalyzes the hydrolysis of the N-glycosidic bond between the uracil (dU) base and the deoxyribose moiety in uracil-containing DNA strands, thereby releasing free uracil. It works on single- or double-stranded DNA containing dU, but not RNA or DNA oligomers (6 or fewer bases).UDG is mainly used to eliminate carryover contaminations in PCR as follows Application ： Removal of uracil bases in single-stranded or double-stranded DNA; removal of cross contamination of PCR products; detection of DNA repair and mutagenesis; improve the cloning efficiency of PCR products; improve the mutagenesis efficiency; protein-DNA interaction studies. This product can be used in PCR, qPCR, RT-PCR, RT-qPCR, and one-step RT-PCR, etc.Enzyme storage buffer: 50mM Tris-HCl (pH 7.5), 100mM NaCl, 0.5mM EDTA, 1mM DTT, 0.1% Triton X-100, 50% (v/v) glycerol.10X Heat-labile Cod UDG Buffer: 700mM Tris-HCl, 100mM NaCl, 10mM EDTA, 1mg/ml BSA, pH 8.0 at 25℃.Inactivation or inhibition: Heating at 50℃ for 10 min can completely inactivate the Heat-labile Cod UDG. Source ： Recombinant Uracil-DNA Glycosylase expressed and purified from E. coli.Purity: Free of DNA endonuclease and exonuclease, phosphatase, and RNase.Applications: Removal of uracil bases in single-stranded or double-stranded DNA; removal of cross contamination of PCR products; detection of DNA repair and mutagenesis; improve the cloning efficiency of PCR products; improve the mutagenesis efficiency; protein-DNA interaction studies. This product can be used in PCR, qPCR, RT-PCR, RT-qPCR, and one-step RT-PCR, etc. Enzyme storage buffer ： 50mM Tris-HCl (pH 7.5), 100mM NaCl, 0.5mM EDTA, 1mM DTT, 0.1% Triton X-100, 50% (v/v) glycerol. Inactivation or inhibition ： Heating at 50℃ for 10 min can completely inactivate the Heat-labile Cod UDG. Precautions ： Heat-labile Cod UDG enzyme is active in most PCR reaction buffers, but it is recommended to examine its compatibility with a particular PCR reaction system used for the first time.The dNTP/dUTP is required but not supplied. We recommend using dNTP/dUTP Mixture (2.5mM each/5mM) .Heat-labile Cod UDG should be stored at -80℃ for long-term storage and -20℃ for routine use. Avoid repeated freeze-thaws.When using the Heat-labile Cod UDG enzyme for One-Step RT-PCR or One-Step qRT-PCR, a heat-resistant reverse transcriptase should be utilized and the reverse transcription should be performed at 50-55℃.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. Set up the PCR or RT-PCR reactions as usual, add Heat-labile Cod UDG to the final concentration of 0.01U/μl, and mix well. Usually, it is not necessary to add the UDG buffer.2. Incubate at 25℃ for 5 min, and then proceed to PCR or RT-PCR cycling conditions.

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