Blood PCR Enhancer

血液 PCR 增强剂

Blood PCR Enhancer

B744167-2ml

2ml

2X

-20°C储存

超低温冰袋运输

-20℃保存。

阿拉丁研发的Blood PCR Enhancer (2X)，即耐血PCR增强剂(2X)，是一种使用阿拉丁的D7241 EnzymoPure™ DNA Polymerase (Blood-resistant)或D7243 EnzymoPure™ HF DNA Polymerase (Blood-resistant)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。Blood PCR Enhancer (2X)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含量基因的效果参考图1。 图1.使用Blood PCR Enhancer (2X)扩增高GC含量目的基因的电泳效果图。图A中可见，HRES1基因488bp片段(GC含量81%)和DIP2A基因1185bp片段(GC含量71%)，在添加Blood PCR Enhancer (2X)后可以扩增出单一的非常明亮的特异性条带，但不添加时就不能扩增获得特异性的目的条带。图B中可见，扩增HRES1基因488bp片段(GC含量81%)时，使用阿拉丁生产的本产品能扩增出单一的条带，而使用公司A和B的类似产品主带比较微弱并且杂带很多，使用常见的最终浓度为1.3M的甜菜碱(Betaine)的扩增高GC含量的该片段时效果也欠佳。本产品如果用于50微升的PCR反应体系，足够用于80个反应；如果用于20微升的PCR反应体系，足够用于200个反应。 注意事项 ： 在使用本产品Blood PCR Enhancer (2X)的同时，必须同时使用和耐血DNA聚合酶配套的最终浓度为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耐血DNA聚合酶的说明书进行。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.PCR反应体系的设置可以参考特定的耐血DNA聚合酶进行，仅仅添加反应总体积一半的本试剂盒所提供的Blood 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HemoTaq™ 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HemoTaq™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(4-x)μl(10-y)μl-10X HemoTaq™ PCR Buffer2μl5μl1XBlood PCR Enhancer (2X)10µl25µl1XdNTP (2.5mM each)2μl5μl0.25mM each引物混合物(10μM each)1μl2.5μl0.5μM eachHemoTaq™ DNA Polymerase1μl2.5μl-抗凝全血xμlyμl-总体积20μl50μl注意：(a)模板使用量：作为PCR模板的抗凝血用量一般为PCR反应体系总体积的1-20%，建议起始使用量为5%。如果PCR反应用于检测基因组的DNA片段，可以适当减少抗凝血用量；如果用于检测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D7245 Blood 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。(b)引物浓度：通常引物的终浓度为0.5μM时可获得良好的检测效果，也可以根据情况在0.1-1.0μM范围内调整引物的终浓度。扩增效率不高的情况下，可提高引物的浓度；发生非特异性反应时，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耐血DNA聚合酶的常规参数，同时可以考虑适当延长延伸时间。 Aladdin's Blood PCR Enhancer (2X) is designed to improve the amplification of DNA with high GC content. It can also be used for direct PCR of anticoagulated blood containing heparin, sodium citrate, or EDTA, to significantly improve the amplification efficiency and specificity.This product 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.Please refer to Figure 1 for the effect of this product on the direct amplification of genes with high GC content in EDTA-anticoagulated blood.Figure 1. GC-rich DNA fragements amplified in PCR reactions containing Aladdin's Blood PCR Enhancer (2X) . A. HRES1 fragment (488bp, 81% GC content) and DIP2A fragment (1185bp, 71% GC content) can be amplified after the addition of this product, but cannot be amplified without this product; B. Compared to similar products from Company A and Company B, Aladdin's Blood PCR Enhancer (2X) has better performance in amplifying the 488bp fragment of HRES1 gene, and better than 1.3M Betaine as well.When a PCR reaction volume of 50μl is used, this product is sufficient for 80 reactions. When a reaction volume of 20µl is used, this product is sufficient for 200 reactions. Precautions ： The Blood PCR Enhancer (2X) must be used in conjunction with the 1X PCR Buffer that is compatible with the blood-resistant DNA polymerase.If crystalline precipitates are present in this product after thawing, incubate in a 50℃ water bath for a few moments to dissolve the precipitates completely before use.We recommend using freshly collected anticoagulated blood for PCR. 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 ： PCR reaction setup:a. Thaw PCR components at room temperature and mix well prior to use. Keep the HemoTaq™ DNA Polymerase on ice.b. Set up the reaction in a PCR tube on ice as follows:ComponentVolume (μl)Volume(μl)Final Nuclease-free Water(14-x) μl(35-y) μl-10X HemoTaq™ PCR Buffer2μl5μl1XdNTP (2.5mM each)2μl5μl0.25mM eachControl Primer Mix (10μM each)1μl2.5μl0.5μM eachHemoTaq™ DNA Polymerase1μl2.5μl-Anticoagulant Whole Bloodxμlyμl-Total Volume20μl50μl-Note 1: When multiple reactions are required, prepare a master mix including all reagents except template and primer, and then dispense to different PCR tubes. Sometimes, the master mix can also include template and primer.Note 2: The final concentration of each primer is recommended to be 0.2μM, but can be adjusted in the range of 0.1-1.0μM. It can be increased when the amplification efficiency is not high, and can be reduced when non-specific amplification occurs.Note 3: The amount of anticoagulated blood used as PCR template is generally 1-20% of the total volume of the PCR reaction, 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 PCR amplification of GC-rich DNA, either add the Blood PCR Enhancer (2X) ( DNA Polymerase (Blood-resistant)1000TD7241SHemoTaq™ DNA Polymerase (Blood-resistant)200TD7243MHemoTaq™ HF DNA Polymerase (Blood-resistant)1000TD7243SHemoTaq™ HF DNA Polymerase (Blood-resistant)200TD7245Blood PCR Enhancer (2X)2mlD7248MPlantTaq™ DNA Polymerase (Chlorophyll-resistant)1000TD7248SPlantTaq™ DNA Polymerase (Chlorophyll-resistant)200TD7285MEasy-Load™ Blood Direct PCR Master Mix (2X)500TD7285SEasy-Load™ Blood Direct PCR Master Mix (2X)100TD7287MEasy-Load™ Blood Direct PCR Master Mix (HF, 2X)500TD7287SEasy-Load™ Blood Direct PCR Master Mix (HF, 2X)100TD7289MEasy-Load™ Plant Direct PCR Master Mix (2X)500TD7289SEasy-Load™ Plant Direct PCR Master Mix (2X)100T