Multi PCR Enhancer

阿拉丁研发的Mult PCR Enhancer (2X)，是一种使用阿拉丁的D7301 Mult Multiplex PCR Kit时，克服因模板高GC含量所导致的PCR扩增障碍的PCR增强剂。高GC含量DNA片段的扩增一直是分子生物学操作过程中的一个难点，本产品可用于含有肝素、柠檬酸钠以及EDTA等抗凝血的直接PCR反应，从而大大提高了高GC含量DNA模板的扩增效率及扩增的特异性。本产品也可以用于普通PCR时扩增高GC含量的目的片段。Mult PCR Enhancer (2X)还可以适用于商业化的常见的多种耐血DNA聚合酶，如阿拉丁的D7241 EnzymoPure™ DNA Polymerase (Blood-resistant)和D7243 EnzymoPure™ HF DNA Polymerase (Blood-resistant)，NEB的Hemo KlenTaq等。本产品直接扩增EDTA抗凝血中高GC含量基因的效果显著优于常用的甜菜碱、DMSO等的效果。本产品如果用于50μl的PCR反应体系，足够用于80个反应；如果用于20μl的PCR反应体系，足够用于200个反应。 注意事项 ： 在使用本产品 PCR Enhancer (2X)的同时，必须同时使用和 Multiplex PCR Kit配套的最终浓度为1X的PCR Buffer。本产品溶解时如果有结晶状沉淀物，可以在50℃水浴温育片刻促进溶解，并确保完全溶解后使用。请尽量使用新近采集的抗凝血样本进行检测；如果是冻存的样本，尽量避免反复冻融，以免在冻融过程中导致目的基因的断裂和降解。设置PCR反应体系时，抗凝血的推荐用量为PCR体系总体积的5-10%左右，可以直接添加到PCR体系中，无需进行任何额外处理。PCR反应结束之后，建议3000-5000g离心3-5min以沉淀血细胞碎片，便于吸取上清用于电泳分析等。注意：由于抗凝血本身的原因，PCR结束后在PCR管底部会出现透明凝胶状物质，此为正常现象。由于PCR反应非常灵敏，可以扩增目的基因序列超过1000万倍，在操作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以确认是否有待扩增DNA的污染。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.PCR反应参数的设置请参照特定的 Multiplex PCR Kit的说明书进行。通常对于扩增高GC含量的DNA模板，延伸时间可以比常规的延伸时间适当延长一些。2.反应体系的设置可以参考特定的 Multiplex PCR Kit的PCR反应体系进行，仅仅添加反应总体积一半的本试剂盒所提供的 PCR Enhancer (2X)即可。可以参考如下反应体系设置PCR反应：a.融解并混匀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反应体系(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和酶的混合物，然后分装到各PCR反应管内。根据情况，有时混合物中也可以包括引物)： 试剂 20μl体系50μl体系终浓度 Nuclease-Free Water (5-x-0.4n)μl (12.5-x-n)μl - 10X PCR Buffer 2μl 5μl 1X PCR Enhancer (2X) 10μl 25μl - dNTP (2.5mM each) 2μl 5μl 0.25mM each Primer mix (10μM each) 0.4μl×n 1μl×n 0.2μM each Template xμl xμl - Control template and primer mix - - Multiplex™ DNA polymerase 1μl 2.5μl - Total volume 20μl 50μl - 注1：n代表多重PCR时使用的引物对种类的数量，即拟扩增片段种类的数量。注2：关于模板使用量。DNA模板的用量对PCR扩增有很大影响。对于高复杂度的DNA样本，如哺乳动物基因组DNA，推荐在20μl反应体系中使用5ng至0.5μg模板DNA。对于低复杂度的DNA，如λDNA或质粒DNA，推荐在20μl反应体系中使用5pg至5ng的模板DNA。注3：关于PCR模板为抗凝血的情况。PCR模板为抗凝血时，模板的用量一般为PCR反应体系总体积的1-20%，建议起始使用量为5%；如果抗凝血多重PCR反应检测的是基因组的DNA片段，可以适当减少抗凝血用量；如果抗凝血多重PCR反应检测的是血液样品中某种病毒或细菌等微生物的目的DNA片段，建议使用50μl的PCR体系，并使用较大的模板血量。对于高GC含量的PCR扩增，可以使用扩增高GC含量DNA片段的D7303S PCR Enhancer (2X)，或者尝试向PCR体系中加入终浓度1-10% (体积百分比)的DMSO。注4：关于PCR模板为植物样品的情况。对于20μl和50μl PCR反应体系，植物样品的推荐用量分别为0.1-1mm和0.3-3mm直径叶片或类似大小其它比较柔嫩的植物组织。如果模板为植物种子，尽量使用鲜嫩的植物种子。用干净的解剖刀去掉种子外壳，剪下直径约0.5-2mm的组织直接放入PCR管内；若种子太小，如番茄种子，可直接使用1-2粒完整的种子放入PCR管内进行扩增。50μl PCR体系，植物叶片或是种子直径不宜超过3mm，太多模板易造成PCR抑制成分偏多，影响PCR效果。推荐取两份植物组织样品进行平行的PCR实验，以降低取样的不稳定性。为确保取样的均一性，建议使用专用的打孔器或解剖刀进行取样，并注意防止取样过程中的交叉污染。每一次取样，可以用2%的次氯酸钠溶液清洗打孔器或解剖刀。对于高GC含量的PCR扩增，可以尝试向PCR体系中加入终浓度1-10% (体积百分比)的DMSO。注5：关于引物浓度。通常引物的终浓度为0.2μM时可获得良好的PCR扩增效果，但也可以根据情况在0.05-0.4μM范围内调整引物的终浓度。扩增效率低的情况下，可提高引物浓度；发生非特异性扩增时，可降低引物浓度。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)。e.把设置好的PCR反应体系置于PCR仪上，开始PCR反应。PCR反应参数的设置可以参考该 Multiplex PCR Kit的常规参数，同时可以考虑适当延长延伸时间。 Aladdin's Mult PCR Enhancer (2X) is a PCR enhancer designed to overcome the barriers to PCR amplification caused by the high GC content of templates when using Mult Multiplex PCR Kit . The amplification of DNA fragments with high GC content has been one of the difficulties in molecular biology, and this product can be used in direct PCR reactions containing anticoagulants such as heparin, sodium citrate, and EDTA, to improve the amplification efficiency and specificity of DNA templates with high GC content. It can also be used in routine PCR to amplify target fragments with high GC content.The Mult PCR Enhancer (2X) is compatible with a variety of commercial blood-resistant DNA polymerases, such as Aladdin's EnzymoPure™ DNA Polymerase (Blood-resistant) , EnzymoPure™ HF DNA Polymerase (Blood-resistant) , and NEB's Hemo KlenTaq, etc.This product is significantly better than betaine and DMSO in improving the PCR amplification of GC-rich genes from EDTA anticoagulated blood.This product is sufficient for 80 PCR reactions of 50μl or 200 PCR reactions of 20μl . Precautions ： The Multi PCR Enhancer (2X) must be used in conjunction with the 1X PCR Buffer included in Multi Multiplex PCR Kit.If crystalline precipitates are present in this product after thawing, incubate it in a 50℃ water bath for a few moments to dissolve the precipitates completely before use.We recommend using the freshly collected anticoagulated blood in PCR reactions. Repeated freeze-thaw of frozen samples that may cause fragmentation and degradation of target genes should be avoided.The anticoagulated blood can be added directly to the PCR reaction without any additional processing, and the recommended amount is about 5-10% of the total volume of the PCR reaction.After amplification, centrifuge the PCR reaction mix at 3000-5000×g for 3-5min to precipitate the blood cell fractions and then take the supernatant for electrophoresis. It is normal to have transparent gelatinous precipitations at the bottom of the PCR reactions because of the presence of anticoagulants.Because the PCR reaction is extremely sensitive, contamination must be avoided during preparation of the 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. Please follow the instructions of Multiplex PCR Kit to set up PCR running conditions. Usually, for amplification of GC-rich DNA templates, the extension time should be longer than the conventional extension time.2. Set up the PCR reaction as follows:a. Thaw the reagents required for PCR reaction setup. Mix well and keep the DNA polymerase on ice until use.b. Set up the PCR reaction on ice. When multiple PCR reactions are required, a master mix can be prepared and dispensed into each PCR reaction tube.ComponentVolume (μl)Volume (μl)Final Nuclease-free Water(5-x-0.4n)μl(12.5-x-n)μl-10X PCR Buffer2μl5μl1X PCR Enhancer (2X)10μl25μl-dNTP (2.5mM each)2μl5μl0.25mM eachPrimer Mix (10μM each)0.4μl×n1μl×n0.2μM eachTemplatexμlxμl-Control Template and Primer Mix---Multiplex™ DNA Polymerase1μl2.5μl-Total Volume20μl50μl-Note 1: n represents the number of primer pairs used in multiplex PCR reaction (i.e., the number of target DNA).Note 2: The amount of DNA template has significant influence on PCR amplification. For high complexity DNA samples, such as mammalian genomic DNA, it is recommended to use 5ng to 0.5μg in a 20μl PCR reaction. For low complexity DNA, such as lambda DNA or plasmid DNA, it is recommended to use 5pg to 5ng in a 20μl reaction.Note 3: For blood samples: The amount of anticoagulated blood used in PCR reactions is generally 1-20% of the total volume, and the recommended starting amount is 5%. For detecting genomic DNA fragment in blood, lower blood concentration can be used. To detect the viral, bacterial or other microbial DNA in blood samples, it is recommended to use a 50μl PCR reaction and use a larger amount of blood. For amplification of GC-rich DNA fragments, either add the PCR Enhancer (2X) or supplement 1-10% DMSO in PCR reactions. Note 4: For plant samples: The recommended plant sample for 20μl and 50μl PCR reactions is 0.1-1mm and 0.3-3mm diameter leaves or other tender plant tissues of similar size, respectively. Fresh and tender seeds can be added directly in PCR reactions, but not dry and hards seeds. Use a clean scalpel to remove the seed shell, cut out 0.5-2mm diameter tissue and put it directly into the PCR reacion. One to two small seeds such as tomato seeds, can be added directly into PCR reactions for DNA amplification. In 50μl of PCR reactions, the diameter of plant leaf or seed should not exceed 3mm, as too many PCR inhibitors present in plant samples will inhibit PCR amplification. It is recommended to perform two parallel PCR reactions for the same sample to reduce variations of sampling. To ensure the uniformity of sampling, it is recommended to use a specialized punch or scalpel for sampling, and to prevent cross-contamination by cleaning the punch or scalpel with 2% sodium hypochlorite solution between each sampling. To amplify GC-rich DNA fragments, supplement 1-10% DMSO in PCR reaction can be attempted.Note 5: The final concentration of each primer in PCR reactions is recommended to be 0.2μM, but can be adjusted in the range of 0.05-0.4μM. Increase the primer concentration when amplification efficiency is low and decrease the primer concentration when non-specific PCR products are amplified.c. Mix well gently by vortex or pipetting. Centrifuge briefly at room temperature to collect the liquid at the bottom of the tube.d. (Optional) When using a thermocycler without a hot lid, add a drop of mineral oil to the reaction to avoid evaporation. e. Transfer the PCR reaction to a thermal cycler and run thermocycling conditions as recommended for the Multiplex PCR Kit. The extension time can be prolonged.

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