UltraBio™ Probe qPCR Mix

UltraBio™ 探针 qPCR 混合物

UltraBio™ Probe qPCR Mix

P751570-1ml

1ml

2X

避光,-20°C储存

超低温冰袋运输

-20℃避光保存，一年有效； 4℃避光保存，一个月内有效。尽量避免反复冻融。

阿拉丁的UltraBio™ Probe qPCR Mix (2X)是一种基于TaqMan等探针的用于实时荧光定量PCR，即探针法qPCR(Quantitative PCR)或real-time PCR的高品质预混液，主要用于cDNA和基因组DNA等的特异性超高灵敏度定量检测。由于使用的探针一般是TaqMan探针，所以本方法也常称作TaqMan探针法。本产品特别适合用于低丰度或高特异性的目的基因的定量或定性检测。例如一些低丰度的mRNA、lncRNA、小RNA、微生物RNA反转录后的高灵敏度检测，一些相似度较高的常规染料法难以区分的同源基因或者基因的SNP检测。检测原理：探针法qPCR不使用荧光染料如SYBR Green等对PCR产物进行荧光染色，而是采用了荧光基团和淬灭基团(quencher)标记的DNA探针靶向拟通过PCR检测的目标序列(设计的探针结合位点通常位于两条引物结合位点之间)。正常情况下，探针上的淬灭基团由于空间上的荧光共振能力转移(FRET)而导致荧光基团淬灭。在PCR反应扩增目标序列时，引物和探针都会退火到目标基因上，随着引物的延伸，Taq酶的5'→3'外切酶活性会导致结合在目标序列上的探针从5’端开始逐渐被降解。探针的荧光基团和淬灭基团被Taq酶切开后，淬灭基团的作用消失，荧光基团就能正常地被激发光所激发而产生荧光了。每经过一个PCR循环，就会有更多的荧光基团被释放，荧光强度与新合成的目标片段数量成正比，从而就可以实现定量检测了。探针通常是目标序列特异性的一段线性DNA，5'端标记FAM或HEX等荧光基团，3'端标记BHQ1、TAMRA或MGB等荧光淬灭基团。本产品的特异性好、灵敏度高：特异性并不仅仅依赖于PCR引物，还依赖于探针的特异性，探针和目标基因发生特异性的结合和降解，才能生成荧光信号，检测灵敏度和特异性通常要显著高于使用SYBR Green等荧光染料的方法。本产品可用于多重检测：单个反应孔中，不同基因对应不同探针，不同探针对应不同荧光标记，即可进行多重荧光定量PCR检测。经测试，在适当优化引物和探针后，本产品可同时用于2-3个基因的检测。本产品使用的UltraBio™ Taq DNA Polymerase是一种与抗体结合的高品质热启动酶，能够实现便捷高效的热启动。UltraBio™ Taq DNA Polymerase中的Taq酶与抗Taq酶的单克隆抗体相互结合，从而抑制了Taq酶的DNA聚合酶活性，这样可以有效避免在低温条件下由引物和模板DNA非特异性退火或引物二聚体引起的非特异性扩增。在PCR反应的预变性步骤中抗体会被加热失活，这样可以确保仅在预变性后才会把Taq酶的活性释放出来，预变性之前不会发生DNA聚合反应，从而大大提高了PCR反应的特异性、灵敏度和定量检测的准确性。本产品包含了UltraBio™ Taq DNA Polymerase、PCR Buffer、dNTPs、稳定剂和镁离子等所有的通用组分，使操作更简单、使用更便捷。用户只需自备引物、探针、样品DNA和去离子水即可。本产品不含ROX，适用于无需ROX作为校正染料的荧光定量PCR仪。ROX的作用是用于校正与PCR无关的荧光波动，从而最大限度减少孔间差异。这种差异可能由多种因素引起，如移液误差及样品蒸发等。不同的荧光定量PCR仪对ROX的要求不同，请根据实际所用仪器选择含高浓度ROX (High ROX)、低浓度ROX (Low ROX)或不含ROX的UltraBio™ Probe qPCR Mix (2X)。通常含高浓度ROX的UltraBio™ Probe qPCR Mix (2X, High ROX)也可以用于不需要ROX或需要低浓度ROX的荧光定量PCR仪。常用仪器所需ROX类型请参考如下表格。 本产品如果用于常规的96孔板qPCR检测(建议反应体系为20μl)，每毫升本产品可以进行100次检测；如果用于常规的384孔板qPCR检测(建议反应体系为10μl)，每毫升本产品可以进行200次检测。 注意事项 ： 探针的荧光标记须根据所使用的qPCR仪荧光兼容性来确定。对于需要ROX作为校正染料的荧光定量PCR仪，避免使用ROX标记探针。用于多重检测时，需要适当优化引物和探针，并使用适当的不同荧光基团标记探针，确定效果后再进行多重检测，一般建议不超过三重检测。使用前需确保整管试剂完全融化，上下颠倒轻轻混匀后使用。混匀过程中尽量避免产生气泡。注意引物退火温度，当退火温度＜60℃时，推荐使用三步法PCR扩增。对于超过350bp或者高GC含量的扩增片段，建议增加延伸时间至60秒或者采用三步法以提高扩增效率。经测试，本产品反复冻融10次对使用效果无显著影响。但仍需尽量避免反复冻融本产品，反复冻融可能使产品性能下降。qPCR检测是超高灵敏度的检测，PCR反应设置区域需尽量避免各种可能的待扩增产物的污染。PCR产物宜密封后丢弃，以避免超高浓度的PCR产物污染实验环境。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反应体系的设置：a.融解并混匀PCR反应所需的各种溶液。 Probe qPCR Mix (2X)完全融解并混匀后置于冰浴上或冰盒内。b.参考下表在室温或冰浴上设置PCR反应体系(以96孔板为例)：Reagent Volume for One PCR Reaction (20μl) Probe qPCR Mix (2X)10µlForward and Reverse Primer Mix (3μM each)2μlProbe0.5μlTemplate DNA2μlRNase-Free Water5.5μl注意：(1) 通常引物的终浓度为0.2-0.5μM时可获得良好的检测效果，也可根据情况在0.1-1.0μM范围内调整引物的终浓度。(2) 最佳的探针(probe)浓度，与使用的荧光定量PCR仪、探针种类、荧光标记物质种类有关。实际使用时请参考仪器说明书，或各荧光探针的具体使用要求进行探针浓度的调节。通常探针的浓度宜低于引物的浓度，例如引物终浓度为0.3μM ，则推荐的探针终浓度为0.2μM，可在0.1-0.3μM范围内调整探针的终浓度。(3) 通常DNA模板的量以1-10ng cDNA或10-100ng基因组DNA为参考用量。因不同物种的模板中含有的目的基因拷贝数不同，如有必要，可对模板进行梯度稀释，以确定最佳的模板使用量。RT-PCR反应得到的cDNA直接作为模板时，其添加量不要超过PCR反应总体积的10%。(4) 96孔板的推荐反应体系为20µl，也可以根据实际实验需求，按比例扩大或缩小反应体系。(5) 建议设置不加模板的阴性对照组。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.将设置好的PCR反应管或PCR反应板置于荧光定量PCR仪上，开始PCR反应。2.PCR反应程序：在Real-time PCR反应前进行模板的预变性，通常设定为95℃ 2分钟，复杂或高GC模板适当延长时间至5分钟。本产品中的 Taq DNA Polymerase可以在15秒内可完成至少300bp的扩增，可以满足绝大多数的qPCR实验；对于超过350bp或者高GC含量的扩增子，建议增加延伸时间至60秒或者采用三步法以提高扩增效率。建议采用如下的PCR程序，本程序是以ABI 7900HT荧光定量PCR仪为例：a.预变性：95℃ 2minb.变性：95℃ 15secc.退火/延伸：60℃ 15-30secd.重复步骤b和步骤c，总共40个循环e.使用荧光定量PCR仪提供的软件分析结果三步法只需在退火/延伸后加一步72℃ 30sec，随后重复步骤b、c及增加的这一步骤共40个循环即可。注：以上举例为常规qPCR反应系统，仅供参考。实际反应条件因模板、引物等的结构不同而各异，需根据模板、引物、目的片段的特点设定最佳反应条件，并根据比例放大或缩小反应体系。常见问题：1.荧光定量PCR结果不理想，出现特异性不好或扩增效率不高时，可能是由于以下原因造成：a.引物设计不佳。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。也可以尝试使用有文献报道使用过的引物，或者从阿拉丁订购经过测试的qPCR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，此时宜更换引物。c.PCR反应体系在室温设置时容易导致非特异性条带，但在使用热启动酶时可以有效避免室温操作导致的非特异性条带的产生。但对于一些较难扩增的产物，可以尝试在冰浴上设置PCR反应体系，以进一步减少非特异性的DNA扩增。d.退火温度不佳，需要优化。这种情况下宜更换引物。e.待扩增片段GC含量较高或长度较长，变性不够充分。此时宜更换引物，使待扩增片段的GC含量和长度适中。f.模板量太低，此时宜适当加大模板量。g.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。2.反应条件优化方法：a.引物浓度：通常引物终浓度为0.3μM时可获得良好检测效果，终浓度可以在0.1-1.0μM范围内适当调整。如果希望提高反应特异性，可降低引物浓度；如果希望提高扩增效率，可增加引物的浓度，从而优化反应体系。b.探针浓度：通常探针终浓度为0.2μM时可获得良好检测效果，终浓度可以在0.1-0.5μM范围内适当调整，但不宜超过引物的终浓度。c.退火温度：建议采用两步法PCR，退火温度60℃进行反应。如果希望提高反应特异性，可提高退火温度，以60-64℃作为退火温度的调整范围。在引物Tm值较低而得不到良好的实验结果时，可尝试进行三步法PCR扩增，三步法的退火温度请以56-64℃作为温度设置的参考范围。d.延伸时间：建议采用两步法PCR，延伸15-30秒。对于超过350bp或者高GC含量的扩增子，建议增加延伸时间至60秒或者采用三步法以提高扩增效率。 Aladdin's UltraBio™ Probe qPCR Mix (2X) is a high-quality supermix for probe-based real-time PCR which is also called TaqMan probe-based qPCR when TaqMan probe is used. It is mainly used in gene expression studies and genomics applications, with high specificity and sensitivity.This product is particularly suitable for quantitative or qualitative analysis of target RNAs with low abundance or high specificity, such as lncRNA, sRNA, low abundance mRNA and microbial RNA. It can also be used to differentiate the expression level of SNPs or some homologous genes with high similarity, which usually could not be achieved using the traditional dye method.The specificity and sensitivity of this kit are superior to that of SYBR Green qPCR Mix. A fluorescent signal is generated by the specific binding with target DNA and the following degradation of the probe. Therefore, the specificity of PCR amplification is ensured by both PCR primers and the probe.This product can be used for multiplex PCR to detect the expression level of multiple genes in a single reaction tube. Different probes with different fluorescence representing different genes are added in a single reaction mixture. Using this kit, two to three genes can be quantified simultaneously in a single reaction mixture with optimized primers and probes.The probe-based real-time PCR uses an oligonucleotide probe complementary to an internal segment of the amplified target DNA. The probe is labeled with two fluorescent moieties. The emission spectrum of one overlaps the excitation spectrum of the other, resulting in the “quenching” of the first fluorophore by the second. During the PCR amplification, both the primers and probes are annealed to the target DNA. With the extension of the primers, the Taq DNA polymerase with 5'→3' exonuclease activity degrades the oligonucleotide probe gradually from the 5' end, releasing the fluorophore group that can be excited by excitation light to produce fluorescence. During PCR, as the target sequence is amplified, more fluorescent groups will be released. The fluorescence intensity is proportional to the number of PCR amplicons, by which the amount of dsDNA generated by PCR amplification can be quantified by measuring the fluorescence intensity. Generally, the 5' end of an oligonucleotide probe is labeled with fluorophore groups such as FAM or HEX and the 3' end is labeled with fluorescence quenchers such as BHQ1, TAMRA or MGB.This kit contains UltraBio™ Taq DNA Polymerase and its monoclonal antibody that binds with DNA polymerase and inhibits its activity at room temperature, allowing reactions to be set up at room temperature. During the initial DNA denaturation step (reaction temperature ~94°C), the antibody is denatured, releasing the polymerase and allowing DNA synthesis to proceed. The use of a hot-start DNA polymerase prevents nonspecific amplification due to mispriming and/or the formation of primer dimers during PCR assembly at lower temperatures, thus greatly improving the specificity, sensitivity and accuracy of qPCR reactions.UltraBio™ Probe qPCR Mix (2X) is a 2X concentrated solution of Taq DNA Polymerase, dNTPs, and all of the components required for PCR, except probes, DNA template and primers.This product does not contain ROX fluorescent dye and can be used on qPCR instrument that does not require ROX as a reference dye. ROX is usually used as a passive reference fluorescent dye in qPCR for signal normalization. ROX is not essential for qPCR, however, adding it can help reduce variations of fluorescent values in samples induced by pipetting errors, sample evaporation, or other technical problems. The optimal ROX concentration in qPCR reaction is dependent on the type of qPCR instrument. Based on the type of qPCR instrument available, choose High ROX, Low ROX or ROX-free qPCR mix. The UltraBio™ SYBR Green qPCR Mix (2X, High ROX) can also be used on qPCR instruments requiring no or low concentrations of ROX. Refer to the following table for qPCR instruments with different requirements of ROX.ROX RequirementReal-time PCR InstrumentNo RequirementBio-Rad Precautions ： PrecautionsPrecautions should be taken to avoid cross-contamination. Discard PCR products after sealing to avoid their contamination of experimental surroundings.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 qPCR reactions:a. Thaw and mix well the Probe qPCR Mix (2X). Once melted, keep it on ice.b. Assemble the qPCR reaction on ice or at room temperature as follows (e.g., in a 96-well plate):ComponentVolume (µl) Probe qPCR Mix (2X)10 µlForward and Reverse Primer Mix (3μM each)2 µlProbe0.5 µlTemplate DNA2 µlRNase-free Waterto 50 µlNote 1: Each primer at a final concentration of 0.2-0.5μM normally works well, but the primer concentration can be optimized between 0.1-1.0 μM. Note 2: The optimal concentration of the probe is dependent on the type of qPCR instrument, probe, and the fluorophore group. In general, the concentration of the probe should be lower than the primer concentration. For example, if the primer concentration is 0.3 μM, the concentration of the probe is recommended to be 0.2 μM, which can be adjusted appropriately in the range of 0.1-0.3 μM.Note 3: Add 1-10ng cDNA or 10-100ng genomic DNA in 20 µl of qPCR reaction as a starting point. When necessary, the DNA templates can be diluted in a gradient manner to determine the optimal amount of DNA template for qPCR reaction. When the cDNA obtained by RT-PCR reaction is directly used as the template, the addition amount should not exceed 10% of the total volume of PCR reaction. Note 4: A reaction volume of 20µl is recommended for each well of a 96-well plate, which can be adjusted based on your requirements.Note 5: Negative control without DNA template is always recommended.c.Mix well reactions by gentle vortex or pipetting. Centrifuge briefly to collect the liquid at the bottom of the PCR tube.2. Transfer qPCR reactions to a qPCR instrument and run thermocycling conditions as follows: StepTemperatureDurationCyclesInitial Denaturation95℃2 min1Denaturation95℃15 sec40Annealing and Extension60℃15-30 secNote 1: The PCR running conditions listed above are for general use only. They can be adjusted based on the template, primer sequence, the length of PCR product or GC content, etc. Note 2: The duration for initial denaturation is usually 2 min, but can be adjusted to 5 min for the amplification of complex or GC-rich DNA fragments.Note 3: The Taq DNA Polymerase is capable of amplifying at least 300bp in 15 seconds. For amplicons longer than 350bp or with high GC content, it is recommended to increase the extension time to 60 s or to use a three-step method to improve amplification efficiency.Note 4: When the annealing temperature of primers is lower than 60ºC, we recommend using the three-step method for PCR amplification.3. qPCR data analysis using the software provided in the qPCR instrument.FAQ:1. Nonspecificity or low amplification efficiency.a. Primer sequence is not well designed. Use primer design tools for primer design to avoid inappropriate GC content, secondary structure, dimer, annealing temperature, length, specificity and other possible problems. Use primers published in the literature or order the tested qPCR primers from .b. The targeted DNA fragments may have a high GC content. It is difficult to amplify DNA fragments with high GC content. Redesign primers at a different region of the DNA target.c. Preparation of PCR reaction mixture at room temperature results in nonspecific amplification easily, which can be prevented by using the hot-start DNA polymerase. To amplify DNA fragment which is hard to be amplified, assembling the PCR reaction mixture on ice can further reduce the nonspecific amplification.d. Annealing temperature needs to be optimized or redesign primers.e. The denaturation is insufficient for DNA amplicons with high GC content or longer length. In this case, redesign primers on regions easier to be amplified.f. The amount of DNA templates is too low. Add more DNA templates.g. DNA templates contain substances that inhibit PCR reactions and can be purified using appropriate DNA purification methods such as column purification.2. Optimization of PCR conditions.a. Primers at a final concentration of 0.2-0.5μM normally work well, but primer concentration can be adjusted between 0.1-1.0 μM. Reduce primer concentration to improve the specificity of PCR amplification and increase primer concentration to improve the amplification efficiency.b. Annealing temperature: Two-step PCR amplification is regularly used with annealing and extension temperature at 60ºC. The annealing/extension temperature can be adjusted in the range of 60-64ºC. The specificity of qPCR reactions can be improved by increasing the annealing temperature appropriately. When the Tm value of primers is low, three-step PCR amplification can be tried. The annealing temperature of the three-step method is recommended to be between 56-64ºC.c. Extension time: Two-step PCR amplification is recommended with an extension time between 15-30 seconds. For amplicons longer than 350bp or with high GC content, it is recommended to increase the extension time to 60 s or to use a three-step amplification method to improve amplification efficiency.