Uracil-DNA Glycosylase (E. coli)

尿嘧啶-DNA糖基化酶(大肠杆菌)

Uracil-DNA Glycosylase (E. coli)

U751665-5000U

5000U

不含UDG酶活力之外的内切或外切脱氧核糖核酸酶、RNase和磷酸酶活性。

-20°C储存

超低温冰袋运输

-20℃保存，至少一年有效。

阿拉丁生产的Uracil-DNA Glycosylase (E. coli)，也称E. coli Uracil-DNA Glycosylase (UDG)或E. coli Uracil N-Glycosylase (UNG)，即大肠杆菌UDG或UNG，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。活性定义：One unit is defined as the amount of enzyme that catalyzes the release of 60 pmol of uracil per minute from double-stranded, uracilcontaining DNA. Activity is measured by release of 3H]-uracil in a 50 µl reaction containing 0.2 µg DNA (104–105 cpm/µg) in 30 minutes at 37℃.阿拉丁生产的E. coli UDG酶活性鉴定结果可参考图1。图1.阿拉丁和N公司E. coli UDG酶催化水解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E. coli UDG，在20µl体系，分别以5µl含dU碱基的1600bp大小的PCR扩增产物为底物和不同量的(0U, 0.0025U, 0.005U, 0.01U, 0.025U, 0.5U) E. coli UDG在1X E. coli UDG Buffer，37℃孵育30min，然后用1%的琼脂糖凝胶进行电泳检测。如图所示，本产品与N公司相比，具有相当的酶活。M, DNA marker )。 用途 ： 防止PCR产物的交叉污染；单核苷酸多态性检测(single nucleotide polymorphism detection，GMPD)；位点特异性突变；蛋白质与DNA相互作用研究；SNP基因分型；PCR产物的克隆；制备含有单链突出末端的PCR产物或cDNA。 来源 ： 大肠杆菌重组、表达和纯化而获得。 酶储存溶液 ： 10mM Tris-HCl (pH 7.4), 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.1mg/ml BSA, 50% (v/v) glycerol。10X E. coli UDG Buffer：200mM Tris-HCl, 10mM EDTA, 10mM DTT, pH 8.0 at 25℃。 失活或抑制 ： 95℃加热10分钟可以使95% E. coli UDG失活。由于经95℃加热10分钟处理后，其仍保持部分活性，建议加入UDG抑制剂(如来自枯草芽孢杆菌噬菌体PBS2的Ugi蛋白或噬菌体phi29的p56蛋白)进一步抑制其酶活以免其继续降解含有dU的PCR产物。 注意事项 ： E. coli UDG酶在大多数PCR反应缓冲液体系中均有活性，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)。由E. coli UDG酶消化产生的DNA链的无碱基位点可通过加热，碱处理或核酸内切核酸酶处理而除去。通常PCR反应过程中的加热步骤可以确保UDG酶消化的位点被完全剪切开。E. coli UDG酶在比较宽泛的pH范围内具有活性，其最适pH值为8.0，E. coli UDG酶活不需要二价阳离子，并被高离子强度(＞ 200 mM)所抑制。E. coli UDG酶可以在PCR反应前清除不慎污染的含dUTP的PCR产物，从而避免由于污染导致的PCR假阳性结果。E. coli UDG在加热变性后可能由于重折叠而在较低温度下表现出残留活性。因此，建议在退火步骤中使用55℃或更高的温度进行后续PCR。E. coli UDG可以用于DNA或cDNA的常规PCR或qPCR扩增体系，但通常不建议用于RT-PCR体系。因为在反转录条件下，通常E. coli UDG会保持活性，并可能消化新合成的cDNA。E. coli UDG酶经95℃加热10min处理后，仍会保持少量活性，如果希望用于RT-PCR体系，需要反转录和PCR分开进行，在反转录时不使用dUTP，在反转录后加入E. coli UDG酶处理，然后进行常规的PCR或qPCR，或者建议加入UDG抑制剂(如来自枯草芽孢杆菌噬菌体PBS2的Ugi蛋白或噬菌体phi29的p56蛋白)进一步抑制E. coli UDG的酶活性。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 ： 1.参考下表设置PCR反应体系，或者参考所使用的PCR扩增体系设置PCR体系，并加入E. coli UDG酶至终浓度为0.01U/μl。通常仅加入PCR buffer即可，无需加入UDG的buffer。 Reagent Volume Volume Final Concentration Nuclease-Free Water (18.325-x)μl (36.65-y)μl - 10X PCR Buffer (with Mg2+) 2.5μl 5μl 1X(1.5mMMg2+) dNTP/dUTP (2.5mMeach/5mM) 2μl 4μl 0.2mMeach/0.4mM Primer mix (10μM each) 2μl 4μl 0.8μM Template xμl yμl 10pg-1μg Taq DNA Polymerase (5U/μl) 0.125μl 0.25μl - E. coliUDG (5U/µl) 0.05μl 0.1μl - Total volume 25μl 50μl - 注1：根据实验需要，dNTP/dUTP (可购买阿拉丁D7376 dNTP/dUTP Mixture (2.5mM each/5mM))的终浓度可在0.2-0.6mM之间调整。镁离子的最终终浓度可在1.0-4.0mM之间调整。注2：对于25μl的PCR反应体系，E. coli UDG (5U/µl)的使用量一般为0.25-0.5U。注3：模板和引物的用量请参考阿拉丁Taq DNA Polymerase 使用说明或相应的PCR体系的产品说明书。2.参考上述反应体系，加入E. coli UDG后混匀，37℃孵育10min (本步骤可以有效去除可能的之前含dUTP的PCR扩增产物的污染)，后续就可以立即进入PCR扩增程序(须确保退火温度不低于55℃)。根据我们的实际测试结果发现，在退火温度不低于55℃的情况下，使用本产品的情况下不会影响PCR扩增的产物量。 Aladdin's Uracil-DNA Glycosylase (E. coli), also known as E. coli UDG enzyme, 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, but not RNA or DNA oligomers (6 or fewer bases).UDG is mainly used to eliminate carryover contaminations in PCR as follows Application ： Prevention of cross-contamination of PCR products; single nucleotide polymorphism detection; site-specific mutagenesis; protein-DNA interaction studies; SNP genotyping; cloning of PCR products; preparation of cDNAs or PCR products with protruding ends.Enzyme storage buffer: 10mM Tris-HCl (pH 7.4), 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.1mg/ml BSA, 50% (v/v) glycerol.10X E. coli UDG Buffer: 200mM Tris-HCl, 10mM EDTA, 10mM DTT, pH 8.0 at 25℃.Inactivation or inhibition: 95% E. coli UDG can be inactivated by heating at 95℃ for 10 min. Since this product remains partially active after heat inactivation, it is recommended to add UDG inhibitors (e.g., Ugi protein from Bacillus subtilis phage PBS2 or p56 protein from phage phi29) to further inhibit its enzyme activity. Source ： Recombinant Uracil-DNA Glycosylase expressed and purified from E. coli.Purity: Free of DNA endonuclease and exonuclease, phosphatase, and RNase.Applications: Prevention of cross-contamination of PCR products; single nucleotide polymorphism detection; site-specific mutagenesis; protein-DNA interaction studies; SNP genotyping; cloning of PCR products; preparation of cDNAs or PCR products with protruding ends. Enzyme storage buffer ： 10mM Tris-HCl (pH 7.4), 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.1mg/ml BSA, 50% (v/v) glycerol. Inactivation or inhibition ： 95% E. coli UDG can be inactivated by heating at 95℃ for 10 min. Since this product remains partially active after heat inactivation, it is recommended to add UDG inhibitors (e.g., Ugi protein from Bacillus subtilis phage PBS2 or p56 protein from phage phi29) to further inhibit its enzyme activity. Precautions ： E. coli 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) .The base free sites of the DNA strand generated by E. coli UDG enzyme can be removed by heating, alkaline treatment or nuclease endonuclease treatment. Usually, the heating step during PCR reactions can make the UDG enzyme digested sites sheared apart.E. coli UDG enzyme is active over a wide pH range, with an optimum pH of 8.0. Its activity does not require divalent cations, but is inhibited by high ionic strength (＞ 200 mM).E. coli UDG enzyme can remove inadvertently contaminated dU-containing PCR products prior to PCR reaction, thus avoiding false positive results for cloning PCR fragments.E. coli UDG enzyme after heat inactivation may remain residual activity at lower temperatures due to refolding. Therefore, the annealing temperature for subsequent PCR should be at least 55℃.E. coli UDG can be used for routine PCR or qPCR with DNA or cDNA template, but is not recommended for RT-PCR reactions, because E. coli UDG is active and may digest the newly synthesized cDNA during the reverse transcription. If it has to be used for RT-PCR, reverse transcription and PCR need to be performed separately: no dUTP during reverse transcription and adding E. coli UDG enzyme after reverse transcription, followed by conventional PCR or qPCR with or without UDG inhibitors (e.g., Ugi protein from Bacillus subtilis phage PBS2 or p56 protein from phage phi29) to further inhibit its enzyme activity.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 reaction as follows, and add the E. coli UDG enzyme to a final concentration of 0.01 U/μl. Usually, it is not necessary to add the UDG buffer.ReagentVolumeVolumeFinal ConcentrationNuclease-Free Water(18.325-x)μl(36.65-y)μl-10X PCR Buffer (with Mg2+)2.5μl5μl1X (1.5mM Mg2+)dNTP/dUTP (2.5mM each/5mM)2μl4μl0.2mM each/0.4mMPrimer Mix (10μM each)2μl4μl0.8μMTemplatexμlyμl10pg-1μgTaq DNA Polymerase (5U/μl)0.125μl0.25μl-E.coli UDG (5U/µl)0.05μl0.1μl-Total Volume25μl 50μl -Note 1: The final concentration of dNTP/dUTP can be adjusted between 0.2-0.6mM, depending on the experimental requirements. The final concentration of magnesium can be optimized between 1.0-4.0mM.Note 2: For a 25 µl PCR reaction, use 0.25-0.5U of E.coli UDG (5U/µl) generally.Note 3: For the amount of template and primer, please refer to the instruction for 's Taq DNA Polymerase or other Taq DNA Polymerases.2. Mix well, incubate at 37℃ for 10 min, and then immediately proceed to PCR cycling conditions (the annealing temperature should be at least 55℃).

One unit is defined as the amount of enzyme that catalyzes the release of 60 pmol of uracil per minute from double-stranded, uracilcontaining DNA. Activity is measured by release of [3H]-uracil in a 50 μl reaction containing 0.2 μg DNA (104–105 cpm/μg) in