EnzymoPure™ DNA Polymerase

EnzymoPure™ DNA聚合酶

EnzymoPure™ DNA Polymerase

D745686-200U

200U

EnzymoPure™

不含DNA内切酶、外切酶和磷酸酯酶，不含RNA酶，满足常规PCR反应要求。

-20°C储存

超低温冰袋运输

-20℃保存。

阿拉丁生产的EnzymoPure™ DNA Polymerase，是一种以嗜热古细菌DNA聚合酶(hyperthermophilic archaeonPyrococcus-like DNA polymerase)为基础通过突变等改造而获得的超高性能DNA聚合酶，它具有扩增速度快、保真度极高、扩增片段可以轻松达到12kb等优点。EnzymoPure™ DNA Polymerase扩增速度极快，扩增小于6kb的DNA片段时，延伸1kb只需要15秒(参考表1)。普通的DNA聚合酶延伸1kb通常需要1-2分钟。EnzymoPure™ DNA polymerase由于其超快的扩增速度，可以显著缩短PCR扩增所需的时间。EnzymoPure™ DNA Polymerase是一种高保真DNA聚合酶', "。EnzymoPure™ DNA Polymerase不仅可以非常高效地催化5'至3'方向的依赖于DNA模板的脱氧核苷酸的聚合反应，它同时还具有3'至5'的外切酶活性(proofreading activity)，它的错误发生概率比Taq酶要低52倍，比pfu酶要约低6倍(参考表1)。"表1. EnzymoPure™ DNA polymerase的主要性能与Taq酶及同类产品的比较。 EnzymoPure™ DNA Polymerase的扩增长度长，可以达到12kb。EnzymoPure™ DNA Polymerase非常适合长片段DNA的扩增，扩增出来的片段可以轻松达到12kb(参考图1和表1)，更长片段的扩增效果未经测试。EnzymoPure™ DNA聚合酶可以轻松胜任具有复杂二级结构的、非常长的DNA片段的准确扩增，因此它是目的基因扩增和克隆的理想选择。 图1. EnzymoPure™ DNA polymerase以λDNA为模板，PCR扩增不同长度(0.5-12kb)片段的的电泳图。 用途 ： 高保真PCR (high fidelity PCR)、点突变、PCR克隆等。 DNA polymerase扩增出来的DNA片段', "为双平端(参考表1)，可以直接用于双平端克隆，但不能直接用于常规的T载体克隆。如需用于T载体克隆，需在 DNA polymerase PCR扩增结束后加入常规量的普通Taq酶在72°C反应5-10分钟，使每条链的3'端加上一个A以形成粘端。", ' 来源 ： DNA polymerase通过大肠杆菌重组、表达、纯化而获得。 酶储存溶液 ： 20mM Tris-HCl (pH 7.5), 1mM DTT, 0.1mM EDTA, 100mM KCl, 200μg/ml BSA and 50% (v/v)glycerol。 失活或抑制 ： 酚氯仿抽提可以使 DNA polymerase失活。 注意事项 ： PCR反应非常灵敏，在进行扩增反应时请注意避免微量待扩增DNA的污染，并尽量考虑设置不加模板的空白对照。'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。\t'], 使用说明 ： 1.PCR反应体系的设置： a.溶解并混匀PCR反应所需的各种溶液。将 DNA polymerase置于冰浴上或冰盒内。b.参考下表在冰浴上设置PCR反应(如果有多个类似的PCR反应，可以先配制大体积的包含水、buffer、dNTP和 DNA polymerase的混合物，然后分装到各PCR反应管内。根据情况，有时混合物中可以包括引物)：扩增片段小于6kb 扩增片段大于6kb 试剂 最终浓度 体积 最终浓度 体积 双蒸水或Milli-Q水-(38-x)μl-(32-y)μl10X Buffer(with Mg2+)1X5μl1X5μldNTP (2.5mM each)0.25mM each5μl 0.5mM each10μl 模板DNA10pg-1μg*xμl10pg-1μg*yμl引物混合物(10μM each)0.2μM each1μl 0.4μM each2μl DNA polymerase2.5U/50μl1μl2.5U/50μl1μl总体积-50μl-50μl*对于不同类型的模板在50μl反应体积中推荐用量如下：哺乳动物基因组DNA：100ng；大肠杆菌基因组DNA：100ng；质粒DNA：5-30ng。扩增大于6kb的片段时，模板量宜适当加大，但过多的模板DNA也容易导致非特异性的PCR产物。注意：参考上表，在扩增大于6kb的片段时dNTP和引物的用量比扩增小于6kb片段时加倍。c.用移液器轻轻吹打混匀或轻微Vortex混匀，室温离心数秒，使液体积聚于管底。d.如果所使用的PCR仪有热盖则省略本步骤。如果PCR仪没有热盖，则在管内滴入一滴矿物油(mineral oil, ST275)。e.各设置好的PCR反应管置于PCR仪上，开始PCR反应。2.PCR反应参数的设置可以参考如下表格： PCR步骤 扩增片段小于6kb时 扩增片段大于6kb时 STEP1(起始变性)92℃ 3min92℃ 3minSTEP2(变性)92℃ 30sec92℃ 30secSTEP3(退火)55℃ 30sec55℃ 30secSTEP4(延伸)68℃ 15s/kb 68℃ 1min/kb STEP5(循环)Go To STEP2 for 30 cyclesGo To STEP2 for 30 cyclesSTEP6(最终延伸)68℃ 10min 68℃ 15min STEP7(临时保存)4℃ forever4℃ forevera.起始变性和变性的温度设置为94℃，延伸温度设置为72℃，其余条件不变时，都可以进行正常的PCR扩增，但扩增效果会稍有下降。对于较难扩增的序列，推荐使用92℃变性和72℃延长。b.PCR反应的设置需根据模板、引物、PCR产物的长度和GC含量等条件的不同设定不同的温度、时间和循环数等。c.STEP4(延伸)的时间设置需根据PCR产物的长度进行设置，对于本产品扩增小于6kb的DNA片段时，推荐每kb产物的延伸时间为15秒。例如PCR产物的长度为1kb，则延伸时间可以设置为15秒，PCR产物的长度为2kb，则延伸时间可以设置为30秒，以此类推。扩增大于6kb的DNA片段时，推荐每kb产物的延伸时间为1分钟。例如PCR产物的长度为10kb，则延伸时间可以设置为10分钟，PCR产物的长度为12kb，则延伸时间可以设置为12分钟，以此类推。d.对于初次进行的PCR，为尽量确保可以扩增出预期的PCR产物，可以把循环数设置为35。对于需进行半定量或定量的PCR反应，循环数一定要进行适当优化，使PCR反应没有达到平台期。常见问题： 1.PCR产物非常少或没有特异性条带。 a.引物设计不佳是PCR过程中最常见的问题。请选择适当的引物设计软件进行引物设计，注意引物的GC含量、二级结构、二聚体、退火温度、长度、特异性等方面的问题。在加入酶切位点等的引物中，一定要注意加入酶切位点等后整条引物的GC含量、二级结构、二聚体、退火温度、长度、特异性等方面的问题。在原有引物效果不佳的情况并且阳性对照引物可以正常工作的情况下，可以考虑更换引物。b.待扩增片段GC含量偏高。GC含量较高的情况下PCR会变得相对比较困难，此时可以使用适合扩增高GC含量DNA片段的GC-rich buffer，并相应地根据GC-rich buffer的要求或说明调整PCR反应参数的设置。直接添加1-10%DMSO或5-20%甘油对于扩增高GC含量的片段也有帮助。c.PCR反应设置时在室温进行容易导致非特异性条件。推荐在冰浴上设置PCR反应。d.由于引物存在一定的二级结构或存在一定的引物二聚体，或引物偏短，导致退火效果不佳。此时可以采用Touch down等方法进行退火，通常采用从65℃逐步缓慢降温到55℃或50℃的方法，使退火更加充分。e.退火温度不佳，需要优化。如果有温度梯度PCR仪，则可以设置退火的温度梯度，摸索退火的最佳温度。如果没有温度梯度PCR仪，则可以通过多次PCR反应摸索最佳的退火温度。f.延伸时间不足。可以在推荐的延伸时间基础上把延伸时间延长2-5倍，对于较难扩增的片段可以设置为每1kb延伸5分钟。g.待扩增片段GC含量较高或长度较长，变性不够充分。可以调节起始变性条件至95℃ 1min甚至95℃ 2-4min。h.在不同PCR仪上进行PCR反应，避免有时PCR仪出现问题。i.循环数不足，适当延长PCR的循环数。通常循环数最高不必超过40，常用的循环数范围为25-35。j.模板含量太低，适当加大模板量，或采用巢式PCR(nested PCR)或二次PCR。巢式PCR即为在原先设计的PCR引物内侧再设计一对PCR引物，然后对第一次PCR产物进行稀释后再进行一次PCR扩增，这样一方面可以起到扩增作用，同时也可以从第一次PCR产物中扩增出特异性条带。二次PCR则为比较简单地用原有引物对第一次PCR产物进行稀释后再进行一次PCR扩增，可以起到扩增作用，但不能去除非特异性条带。k.模板中含有抑制PCR反应的物质，可以用适当的DNA纯化方法例如柱纯化等纯化模板DNA。l.对PCR引物进行脱盐纯化。m.使用高质量的dNTP混合物。n.适当增加DNA polymerase的用量。o.当产生较多非特异性条带时，可以适当提高退火温度。p.注意设置适当的阳性对照和阴性对照通常会有很大帮助。2.杂带较多或条带弥散。 a.退火温度提高2-5°C。b.减少DNA模板的用量。c.PCR反应设置时在室温进行容易产生非特异性条带。推荐在冰浴上设置PCR反应。d.适当减少 DNA polymerase用量。e.适当缩短延伸时间。 One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 74℃. Application ： high-fidelity PCR, point mutation, and gene cloning, etc. The amplified PCR products by DNA polymerase are blunt-ended and can be used directly for blunt-end cloning. Prior to be cloned into T vectors, the PCR products generated by the DNA polymerase must be dA-tailed by incubation with a conventional Taq DNA polymerase at 72℃ for 5-10 minutes.Definition of activity: One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 74℃.Purity: free of DNA endonuclease and exonuclease, phosphatase, and RNAase.Enzyme storage buffer: 20mM Tris-HCl (pH 7.5), 1mM DTT, 0.1mM EDTA, 100mM KCl, 200µg/ml BSA and 50% (v/v) glycerol.Inactivation or inhibition: DNA polymerase can be deactivated by phenol chloroform extraction. Source ： Recombinant hyperthermophilic archaeon Pyrococcus-like DNA polymerase expressed in E. coli. Enzyme storage buffer ： 20mM Tris-HCl (pH 7.5), 1mM DTT, 0.1mM EDTA, 100mM KCl, 200µg/ml BSA and 50% (v/v) glycerol. Inactivation or inhibition ： DNA polymerase can be deactivated by phenol chloroform extraction. Precautions ： Because PCR reaction is extremely sensitive, contamination must be avoided during preparation of PCR reactions. Negative control without templates is recommended for all PCR assays to control contamination.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.Prepare PCR reactions:a.Thaw PCR components at room temperature and mix well prior to use. Keep the DNA polymerase on ice.b.Set up the PCR reaction on ice as follows: To amplify dsDNA ＜ 6kbTo amplify dsDNA ＞ 6kbComponentFinal ConcentrationVolumeFinal ConcentrationVolumeNuclease-free water-(36.5-x)μl-(29-y)μl10X Buffer (with Mg2+)1×5μl1×5μldNTP (2.5mM each)0.25mM each5μl0.5mM each10μlTemplate DNA10pg-1μg*xμl10pg-1μg*yμlPrimer mixtures (10μM each)0.2μM each1μl0.4μM each2μl DNA Polymerase2.5U/50μl1μl2.5U/50μl1μlTotal volume-50μl-50μlNote 1: When multiple reactions are required, prepare a master mix including all reagents except template and primer, and then dispense to different nuclease-free PCR tubes. Sometimes, the master mix can also include template and primer.Note 2: When amplifying fragments larger than 6kb, the amount of template should be increased appropriately, but too much template DNA can also easily lead to non-specific PCR amplification products. * The recommended amount of template varies for different types of DNA. In a reaction volume of 50μl, use 100 ng of Mammalian genomic DNA, 100 ng of E. coli genomic DNA, or 5-30 ng of Plasmid DNA. c.Mix the PCR reaction mixture by gentle vortex or pipetting. Centrifuge briefly to collect liquid at the bottom of the PCR tube. d.(Optional) When using a thermocycler without a heated lid, place a drop of mineral oil onto the top of PCR reaction mixture.2.Transfer the PCR reaction to a thermocycler and run thermocycling conditions as follows:StepTo amplify dsDNA ＜6kbTo amplify dsDNA ＞ 6kbCyclesInitial denaturation94℃ 3min94℃ 3min1Denaturation94℃ 30sec94℃ 30sec30Annealing55℃ 30sec55℃ 30secExtension68℃ 15s/kb68℃ 1min/kbFinal extension68℃ 10min68℃ 15min1Hold4℃ forever4℃ forever-Note 1: The extension temperature can also be set to 72℃, but the amplification will be slightly reduced. For DNA fragments difficult to amplify, denaturation at 92℃ and extension at 72℃ are recommended. Note 2: PCR running conditions should be adjusted based on the template, primer sequence, the length of PCR product or GC content, etc.Note 3: The optimal extension time varies depending on the amplicon length. For amplification of DNA fragments shorter than 6kb, the recommended extension time is 15 seconds per kb (e.g. use 15 seconds to amplify 1kb fragment and 30 seconds to amplify 2kb fragment). When amplifying DNA fragments longer than 6kb, the recommended extension time is 1 minute per kb (e.g., use 5 minutes to amplify 5kb fragment). Note 4: For initial PCR, the number of cycles can be set to 35 to ensure that the expected PCR product can be amplified. The number of cycles for semi-quantitative or quantitative PCR analysis must be optimized appropriately so that the PCR reaction does not reach a plateau.FAQ:1.Few PCR products or no specific bands.a.It could be due to poor design of primers. Use primer design tools for primer design to avoid inappropriate GC content, secondary structure, dimer, annealing temperature, length, specificity and other possible problems. When adding restriction enzyme cutting sites in the primer sequence, the same problems need to be considered. In the case that positive control primers work normally but not your primers, redesign primers.b.DNA to be amplified may have a high GC content. High GC genes are relatively difficult to be amplified. In such a case, GC-rich buffer suitable for amplifying DNA with high GC content can be used, and PCR reaction parameters should be adjusted accordingly. Direct addition of 1-10% DMSO or 5-20% glycerol is also helpful for amplifying fragments with high GC content.c.PCR reactions set up at room temperature tend to produce non-specific bands. It is recommended to set up PCR reactions on ice.d.The presence of secondary structure in primers, primer dimers or short primers, may result in poor annealing of primers to the target sequence. In this case, try touch down or other methods for annealing. A gradual cooling from 65℃ to 55℃ or 50℃ is usually used to make annealing more efficiently. e.The annealing temperature needs to be optimized. If necessary, use a temperature gradient to determine the optimal annealing temperature for each template-primer pair combination. f.Insufficient extension time. The extension time can be extended 2-5 times from the recommended extension time, and can be set to 5 minutes per 1kb for fragments difficult to amplify. g.Insufficient denaturation. To amplify long DNA or high GC DNA fragment, the initial denaturation temperature can be adjusted to 95℃ for 1 min or even 95℃ for 2-4 min.h.Perform PCR reactions on a different thermal cycler to avoid possible problems with the instrument.i.Insufficient number of PCR cycles. Try more PCR cycles, but do not exceed 40 cycles. j.Insufficient amount of template. Add more DNA templates or try nested PCR or secondary PCR. Nested PCR is to design another pair of PCR primers inside the original PCR primers, and then conduct PCR amplification again with the diluted first PCR product as template. Instead, secondary PCR uses the same primers for second PCR amplification with the diluted PCR product as template. Nested PCR usually can remove the non-specific DNA amplification, but secondary PCR cannot.k.DNA sample contains substances that inhibit the PCR reactions. In such a case, template DNA can be purified using appropriate DNA purification methods such as column purification.l.Use high-purity primers.m.Use high-quality dNTP mixture.n.Increase the amount of DNA polymerase appropriately.o.When non-specific products are produced, increase the annealing temperature appropriately.p.Positive and negative controls are always recommended when optimizing PCR reactions.2.Occurrence of non-specific bands or DNA smear when examined by agarose gel electrophoresis.a.Increase the annealing temperature by 2-5℃.b.Reduce the amount of DNA template.c.PCR reactions set up at room temperature tend to produce non-specific bands. It is recommended to set up PCR reactions on ice.d.Reduce the amount of DNA polymerase appropriately.e.Reduce the extension time appropriately.

One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid ', insoluble material in 30 minutes at 74°C., '