UltraBio™ Multiplex PCR Master Mix

阿拉丁生产的UltraBio™ Multiplex PCR Master Mix (2X)，即UltraBio™多重PCR预混液(2X)，是一种使用特别便捷的专门用于多重PCR扩增的2倍浓度的PCR预混液，并且PCR结束后可以直接上样电泳无需添加上样缓冲液。本产品含有Mult DNA polymerase，可以高效、高特异性和高灵敏度的适用于对多个(可以超过20个)目标DNA片段同时进行非常均衡的PCR扩增。并且本产品不仅可以用于普通的多重PCR，更可以用于全血、血清或植物样品的直接PCR，便于直接用于血液样品中细菌和病毒感染、基因突变等的多重PCR检测，或者用于植物样品的基因突变、微生物感染等的多重PCR检测。多重PCR是一种通过单个PCR反应同时对至少两个或多个DNA片段进行扩增的PCR技术。目前该技术已被广泛应用于科学研究、疾病诊断和法庭或诊断性的基因分型(Genotyping)等多个领域。该技术还可以被用于以cDNA为模板的基因定量或半定量的表达分析，其特别适合于各种动植物、真菌、细菌或病毒等微量样品进行多基因检测，具备高特异性和高灵敏度的突出优点。本产品多重扩增性能优越。本产品实测可以轻松实现15个目标DNA片段的同时并且非常均衡的高效、高特异性和高灵敏度扩增(参考图1)。对于低至1pg的模板量，也可以通过仅30个PCR循环而被很好地扩增(参考图1)。普通的PCR试剂盒会由于PCR扩增时引物和模板的偏好性，对于不同的引物和不同的模板会产生不同的扩增效率，导致部分片段容易被扩增，而部分片段较难被扩增。本试剂盒使用了通过精心筛选和突变优化的非常适合用于多重PCR的DNA聚合酶Mult DNA polymerase和反复优化的配套缓冲液，确保可以实现多个目标DNA片段的高效、高特异性和高灵敏度的均衡扩增。本产品可同时对低至100pg的样品模板的15个基因片段进行有效的多重PCR检测(图1)。 图1. 阿拉丁生产的UltraBio™ Multiplex PCR Master Mix (2X)进行多重PCR扩增的实测电泳效果图。以图中指定量的Lambda噬菌体基因组DNA为模板，按照本产品的使用说明每个样品采用20μl PCR扩增体系，使用15对引物进行多重PCR扩增，每条引物的终浓度为0.2μM，PCR扩增条件为：94℃预变性5min；94℃变性30sec、60℃退火30sec、68℃延伸3min；最后68℃延伸10min；共30个PCR循环。PCR结束后，取2μl扩增产物进行电泳检测。扩增的片段从小到大依次为113bp、143bp、199bp、253bp、303bp、406bp、501bp、576bp、710bp、802bp、903bp、989bp、1167bp、1300bp和1463bp。M, DNA marker )。图中可见低至10pg的模板就可以扩增获得15条非常单一明亮的条带。本产品兼容血液和植物样品。全血或血清样品无须进行DNA的提取和纯化，即可直接作为模板用于本试剂盒的多重PCR检测。本产品适用于EDTA、肝素或柠檬酸钠抗凝血样品或干血斑样品。植物样品也可以直接用于本试剂盒的多重PCR检测，完全无须对植物样品进行DNA的提取和纯化。本产品提供了阳性对照，便于验证本产品的效果。本试剂盒提供了Control template and primer mix，预先混合了模板和引物，可以作为阳性对照用于验证确认本产品的多重PCR扩增效果。Control template and primer mix中包含了15对引物，可以扩增出如图1所示的从113bp-1463bp的共15个条带。本产品使用特别便捷。仅需添加引物、模板和水即可进行多重PCR，并且PCR结束后可以直接进行电泳检测，无需添加上样缓冲液。本产品的扩增产物可以用于TA克隆。 "使用本产品获得的PCR产物带有3'-dA overhangs的粘性末端，可直接用于与T载体连接进行TA克隆。本产品中所使用的Mult DNA polymerase的保真性和Taq DNA聚合酶相近，因此本产品主要推荐用于定性和半定量检测。本产品如果用于20µl的PCR反应体系，两种包装的本产品分别足够用于100个和500个反应。 注意事项 ： 引物设计对于多重PCR的成功与否至关重要。引物的设计一方面需要满足常规的引物设计规则，避免出现非特异性扩增和无法扩增，设计好的引物对应逐一通过PCR验证，然后才能选择效果较好的引物对用于多重PCR。并且引物的设计在尽可能的情况下，如果Tm值按照Tm = 2n(A) + 2n(T) + 4n(C) + 4n(C)进行计算(例如一条引物含有3个A、7个T、4个G和6个C，那么Tm = 2X3 + 2X7 + 4X4 + 4X6 = 60)，Tm值不能低于60℃，通常按此计算Tm不低于65℃时用于多重PCR的效果会更佳。推荐使用高质量引物(经脱盐处理、PAGE或HPLC纯化)，建议使用前预先混合所用所有引物对，调整各对引物母液浓度均达到10μM，最终使其在PCR反应体系中终浓度达0.2μM。推荐的延伸速度为2min/kb，对于较难扩增的靶序列，可适当调整延伸时间为3-4min/kb。如果PCR产物条带较多，建议PCR反应后电泳检测的上样量调整为约2μl，过多的上样量容易导致电泳条带不太平整。由于多重PCR反应非常灵敏，在使用本产品时请注意避免微量待扩增DNA的污染，建议设置不加模板的空白对照以确认是否有待扩增DNA的污染。需自备Nuclease-Free Water。推荐选购阿拉丁生产的ST876 Ultrapure Water (DNase/RNase-Free, Sterile)。对模板GC含量过高的情况，推荐使用D7301 Multiplex PCR Kit并和D7303 PCR Enhancer (2X)配套使用。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.引物设计：引物设计对于成功进行多重PCR至关重要，建议使用适当的引物设计软件进行引物设计：a.引物的长度通常为20-30个核苷酸。b.GC含量为40-60% (优选45-55%)。c.避免所用的多个引物的3'末端出现互补序列，避免引物3'末端有3个或更多的G / C，避免引物内的二级结构。d.在尽可能的情况下所用引物的Tm值应不低于60℃，推荐在Tm值在65-68℃之间更佳，引物间的Tm值差异应控制在5-6℃以内。此处提到Tm值按照Tm = 2n(A) + 2n(T) + 4n(C) + 4n(C)进行计算，例如一条引物含有3个A、7个T、4个G和6个C，那么Tm = 2X3 + 2X7 + 4X4 + 4X6 = 60。e.建议扩增的目标片段不超过1500bp。虽然目标片段大约1500bp时也能被很好地扩增，但过长的片段和较短的片段同时进行PCR扩增的时候，相对更容易产生亮度不太均匀的条带。f.建议使用经过脱盐、PAGE或HPLC纯化的引物，并溶于TE buffer (10mM Tris-Cl,1mM EDTA, pH 8.0)。2.引物的配制：每种合成的引物推荐配制为100µM，然后每个引物对1:1混合并加入适量水配制成10µM的引物对。例如，如果合成得到的一个5’端引物A的量是20nmol，另外一个相应的3’端引物B的量是19nmol。在引物A中加入200µl水或TE，配制成浓度为100µM，在引物B中加入190µl水或TE也配制成浓度为100µM。吸取20µl 100µM引物A和20µl 100µM引物B到一新的离心管中，再加入160µl水，混匀后即可得到可以直接用于多重PCR的引物对(10µM each)。3.多重PCR扩增:a.反应体系的设置。融解并混匀Easy-Load™ Multiplex PCR Master Mix (2X)，置于冰浴上或冰盒内。参考下表设置PCR反应体系。可根据情况，将多对引物进行等浓度预混合。 Component SampleControlSampleControlFinal concentration Nuclease-Free Water (10-x-0.4n)μl 5μl (25-x-n)μl 12.5μl - Easy-Load™ Multiplex PCR Master Mix (2X) 10μl 10μl 25μl 25μl 1X Primer mix (10μM each) 0.4μl×n - 1μl×n - 0.2μM each Template xμl - xμl - - Control template and primer mix - 5μl - 12.5μl - Total volume 20μl 20μl 50μl 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，推荐使用D7301 Multiplex PCR Kit并和D7303S PCR Enhancer (2X)配套使用，或者尝试向PCR体系中加入终浓度1-10% (体积百分比)的DMSO。对于干血斑样品，20μl和50μl的PCR体系中分别推荐使用约0.8平方毫米和2平方毫米的干血斑。注4：关于PCR模板为植物样品的情况。PCR模板为抗凝血时，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。b.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。c.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)。d.把设置好的PCR反应体系置于PCR仪上，开始PCR反应。PCR反应的设置可以参考如下表格。 Step CyclesTemperatureTimeDescription 1 - 94℃ 5min Initial denaturation 2 30-40 94℃ 30sec Denaturation 60℃ 30sec Annealing 68℃ 2min/kb Extension 3 - 68℃ 10min Final extension 4 - 4℃ Forever Hold 注1：需根据每次反应的模板、引物、PCR产物的长度和GC含量等适当优化PCR反应条件，包括退火温度、退火时间、循环数等。通常退火温度可在55-60℃范围内适当调整，或者可以对退火温度采用Touch down的方式，确保退火效果良好。注2：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。后续可以根据实际的PCR效果适当调整循环数。4.电泳检测。多重PCR反应结束后，即可进行常规的凝胶电泳检测(对于植物等比较特殊的样品，如有必要，可以离心取上清进行电泳)。阳性对照的检测结果可以参考图1。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。推荐使用D7301 Multiplex PCR Kit并和D7303S PCR Enhancer (2X)配套使用。c.部分目的片段过长。尽管 DNA Polymerase可以扩增最长达5kb的DNA片段，但在多重PCR时，大多数时候更适合扩增1.5kb以下的片段。过长的目的片段不太适合在多重PCR时进行扩增。d.引物的二级结构、引物二聚体或引物偏短会导致退火效果不佳。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。可按照每1kb片段延伸2分钟进行设置，对于较难扩增的片段可以设置为每1kb片段延伸3-4分钟。g.在不同PCR仪上进行PCR反应，避免有时PCR仪出现问题。h.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30-40。i.模板用量偏少，可以适当加大模板用量。j.对PCR引物进行脱盐甚至PAGE胶或HPLC纯化。k.使用高质量的dNTP混合物。l.适当增加 DNA polymerase的用量。m.当产生较多非特异性条带时，可以适当提高退火温度。n.注意设置本产品提供的Control template and primer mix作为阳性对照，以确认整个多重PCR体系可以正常工作。o.确保没有漏加任何PCR反应的组分。2.杂带较多或条带弥散。a.退火温度提高 2-5°C。b.适当检测PCR的循环数。c.适当减少模板的用量。d.在室温配制PCR体系容易产生非特异性条带。推荐在冰浴上配制PCR反应体系。e.适当减少 DNA polymerase的用量，或推荐使用D7301 Multiplex PCR Kit并和D7303S PCR Enhancer (2X)配套使用。f.适当缩短延伸时间。 Aladdin's UltraBio™ Multiplex PCR Master Mix (2X) is designed specially for simultaneous, well-balanced amplification of multiple DNA targets in a single PCR reaction with high efficiency, specificity and sensitivity. It is a 2X concentrated solution of Mult DNA polymerase, dNTPs, and all of the components required for PCR, except DNA template and primers. The premix also contains a 2X loading buffer that enables PCR reactions to be directly loaded on an agarose gel for electrophoresis. This product can be used not only for ordinary multiplex PCR, but also for direct PCR of whole blood, serum or plant samples.ItemComponentQuantityD7305S-1UltraBio™ Multiplex PCR Master Mix (2X)1mlD7305S-2Control Template and Primer Mix25μlManual-1 copyMultiplex PCR technique has been widely used in scientific research, disease diagnosis and diagnostic genotyping, such as quantitative or semi-quantitative gene expression analysis using cDNA as template, multi-gene detection of trace samples of animal, plant, fungi, bacteria or viruses.ItemComponentQuantityD7305M-1UltraBio™ Multiplex PCR Master Mix (2X)1ml×5D7305M-2Control Template and Primer Mix50μlManual-1 copyThis product has superior multiplex amplification performance. This product can easily achieve the simultaneous, well-balanced amplification of 15 target DNA fragments in a single PCR reaction with high efficiency, specificity and sensitivity (Figure 1). Template as low as 1pg can be amplified successfully with only 30 PCR cycles (Figure 1). Regular PCR reagents normally have a bias towards different primers and DNA templates, while the Mult DNA Polymerase has been diligently optimized for multiplex PCR to ensure well-balanced amplification of multiple target DNA fragments with high efficiency, specificity and sensitivity.This product can simultaneously amplify 15 different DNA fragments from sample templates as low as 100pg (Figure 1).Figure 1. Agarose gel electrophoresis of multiplex PCR products from different amounts of Lamda DNA using Aladdin's UltraBio™ Multiplex PCR Master Mix (2X) . Fifteen primer pairs with a final concentration of each primer at 0.2μM are used to amplify DNA fragments of 113bp, 143bp, 199bp, 253bp, 303bp, 406bp, 501bp, 576bp, 710bp, 802bp, 903bp, 989bp, 1167bp, 1300bp and 1463bp, respectively. M, DNA marker . As shown in the figure, 15 different DNA fragments can be amplified from Lamda DNA as low as 10pg.This product is compatible with blood and plant samples. Whole blood, serum or plant samples can be added directly to PCR reactions without DNA extraction and purification. Both dried blood spots on cards and blood with EDTA, heparin or sodium citrate anticoagulants are compatible with this product. Positive control template and primer mix are provided for troubleshooting. The Control Template and Primer Premix contains 15 primer pairs used to amplify DNA fragments with different size ranging from 113bp to 1463bp.This product generates PCR products with 3'-dA overhangs, which can be used for TA cloning subsequently. The Mult DNA polymerase contained in the Master Mix has a fidelity similar to Taq DNA polymerase and is therefore recommended primarily for qualitative and semi-quantitative analysis.s Application ： DNA amplification; multiplex PCR detection of bacterial or viral infection and gene mutation in blood and plant samples.Storage Conditions：Store reagents at -20℃ upon receipt. Avoid repeated freeze-thaws.Precautions：Primer design is the key to a successful multiplex PCR. Primers need to meet the conventional primer rules to avoid non-specific amplification and non-amplification, and should be verified by PCR individually. The melting temperature of all primers for multiplex PCR should be more than 60ºC. Better multiplex PCR results are normally obtained using primers with Tm higher than 65℃. The Tm value is calculated according to the formula: Tm (℃) = 2n(A) + 2n(T) + 4n(G) + 4n(C) (for example, for a primer contains 3 A, 7 T, 4 G and 6 C, Tm = 2×3 + 2×7 + 4×4 + 4×6 = 60℃).Primers should be desalted, PAGE or HPLC-purified. It is recommended to prepare a primer mix solution including forward and reverse primers at 10μM each.An extension time of two minutes per kb is recommended. For DNA targets hard to be amplified, try 3-4 min per kb.For abundant PCR products, it is recommended to load 2μl of PCR reaction for agarose gel electrophoresis analysis. Too many PCR products may lead to uneven DNA bands.Because the multiplex PCR reaction is extremely sensitive, contamination must be avoided during the preparation of PCR reactions. Negative control without templates is recommended for all PCR assays to control contamination.We recommend using ’s Multiplex PCR Kit and PCR Enhancer to amplify GC-rich DNA fragments.Nuclease-free water is not provided in this product. Use the Ultrapure Wateror other types of ultra-pure H2O.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. Precautions ： Primer design is the key to a successful multiplex PCR. Primers need to meet the conventional primer rules to avoid non-specific amplification and non-amplification, and should be verified by PCR individually. The melting temperature of all primers for multiplex PCR should be more than 60ºC. Better multiplex PCR results are normally obtained using primers with Tm higher than 65℃. The Tm value is calculated according to the formula: Tm (℃) = 2n(A) + 2n(T) + 4n(G) + 4n(C) (for example, for a primer contains 3 A, 7 T, 4 G and 6 C, Tm = 2×3 + 2×7 + 4×4 + 4×6 = 60℃).Primers should be desalted, PAGE or HPLC-purified. It is recommended to prepare a primer mix solution including forward and reverse primers at 10μM each.An extension time of two minutes per kb is recommended. For DNA targets hard to be amplified, try 3-4 min per kb.For abundant PCR products, it is recommended to load 2μl of PCR reaction for agarose gel electrophoresis analysis. Too many PCR products may lead to uneven DNA bands.Because the multiplex PCR reaction is extremely sensitive, contamination must be avoided during the preparation of PCR reactions. Negative control without templates is recommended for all PCR assays to control contamination.We recommend using ’s Multi™ Multiplex PCR Kit and Multi™ PCR Enhancer to amplify GC-rich DNA fragments.Nuclease-free water is not provided in this product. Use the Pure™ Ultrapure Water or other types of ultra-pure H2O.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. Primer design:Good primers are crucial for a successful multiplex PCR. We recommend using primer design tools for primer design. Primers should comply with the following rules:a. Primers are generally 20-30 nucleotides in length; b. Primers have a GC content of 40-60% (preferably 50-60%);c. Try to avoid complementary sequences at the 3’-end of all primers, runs of three or more G/C at the 3' end, and secondary structures within primers;d. The melting temperature of all primers for multiplex PCR should be more than 60°C according to the formula: Tm (°C) = 2n(A) + 2n(T) + 4n(G) + 4n(C). Primers with Tm between 65-68°C are preferred and the difference in Tm of primer pairs should be within 5-6℃.e. It is recommended that the target fragment should not exceed 1500bp. Although the target fragment of approximately 1500bp can be well amplified, it is more likely to produce uneven bands when long fragments and short fragments are amplified simultaneously.f. Primers should be desalted, PAGE or HPLC-purified, as the primer quality is a critical factor for good multiplex PCR. Primers should be dissolved in TE buffer (10 mM Tris-HCl, 1.0 mM EDTA, pH 8.0).2. Preparation of primer stock:Prepare primer stock at 100µM in TE buffer for each primer, and then mix each primer pair at a 1:1 ratio in an appropriate amount of ultra-pure H2O to produce a primer pair of 10µM each.3. Preparation of PCR reactions:a. Thaw the Easy-Load™ Multiplex PCR Master Mix (2X) and Control Template and Primer Mix at room temperature. Mix well gently by inversion and centrifuge briefly. Keep the reagents on ice.b. Assemble PCR reactions on ice as follows:ComponentSampleControlSampleControlFinal concentrationNuclease-Free Water(10-x-0.4n)μl5μl(25-x-n)μl12.5μl-Easy-Load™ Multiplex PCR Master Mix (2X)10μl10μl25μl25μl1XPrimer Mix (10μM each)0.4μl×n-1μl×n-0.2μM eachTemplatexμl-xμl--Control Template and Primer Mix-5μl-12.5μl-Total volume20μl20μl50μl50μl-Note: (a) ‘n’ represents the number of primer pairs, that is the number of DNA targets. (b) The amount of DNA template has a significant influence on PCR amplification. For DNA samples with high complexity, such as mammalian genome DNA, 5ng-0.5μg of DNA template is recommended in 20μl of PCR reaction. For low complexity DNA, such as Lamda DNA or plasmid DNA, 5pg-5ng of DNA template is recommended in 20μl of PCR reaction. (c) For blood samples: The recommended blood concentration is 1-20%, and 5% can be used as a starting point. For detecting genomic DNA fragments in blood, lower blood concentration can be used. To detect the viral, bacterial or other microbial DNA in blood samples, a higher blood concentration is recommended. We recommend using Multiplex PCR Kit and PCR Enhancer for the amplification of DNA with high GC content, or adding 1−10% DMSO in PCR reactions. For dried blood spot samples, 0.8 mm2 and 2 mm2 are recommended for 20μl and 50μl of PCR reactions, respectively. (d) For plant samples: When setting up PCR reactions, 0.1-1mm and 0.3-3mm diameter leaves or other tender plant tissues of similar size are recommended for 20μl and 50μl of PCR reactions, respectively. Fresh and tender seeds are suitable for this Master Mix, but not dry and hard seeds. Use a clean scalpel to remove the seed shell, cut out 0.5-2mm diameter tissue, and put it directly into the PCR reaction. 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 introduced by 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 time of sampling. To amplify DNA with high GC content, DMSO can be added to PCR reactions at a final concentration of 1-10% (v/v). (e) Each primer at a final concentration of 0.5 μM normally works well, but primer concentration can be optimized between 0.05-0.4 μM. Increase the primer concentration when amplification efficiency is low and decrease the primer concentration when non-specific PCR products are amplified. c. Mix the reaction by gentle vortex or pipetting. centrifuge briefly to allow liquid to accumulate at the bottom of the PCR 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 reactions to a thermal cycler and run thermocycling conditions as follows:StepTemperatureDurationCyclesInitial Denaturation94℃5 min1Denaturation94℃30 sec30-40Annealing60℃30 secExtension68℃2 min/kbFinal Extension68℃10 min1Holding4℃--Note 1: Optimize PCR running conditions based on the template, primer sequence, amplicon length and GC content, etc. The annealing temperature can be adjusted between 55-60℃. Touch down method can also be applied to make annealing more efficient. Note 2: Use 35 cycles for the first time to ensure the expected DNA fragments are amplified. The cycle number can be adjusted appropriately based on multiplex PCR results. 4. Analyze PCR products by agarose gel electrophoresis: The Master Mix contains premixed loading dye that enables reactions to be loaded directly on agarose gel for electrophoresis. When necessary, centrifuge PCR reactions at 3000-5000×g for 3-5 minutes to collect the supernatant for gel electrophoresis. Refer to Figure 1 for the multiplex PCR results of positive control.FAQ:1. No product at all or low yield.a. Primer sequence is not well designed. Use primer design tools 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 well but not your primers, redesign primers.b. DNA may have a high GC content. High GC genes are relatively difficult to be amplified. We recommend using Multiplex PCR Kit and PCR Enhancer for the amplification of GC-rich DNA fragments.c. Some DNA fragments are too long to be amplified. Although Multiplex DNA polymerase can amplify DNA fragments up to 5 kb in length, it is more suitable for the amplification of DNA fragments less than 1.5 kb in multiplex PCR reactions. Multiplex PCR does not work very well for the amplification of long DNA fragments. d. The presence of secondary structure in primers, primer dimmers or short primers, may result in poor annealing of primers to the target sequence. Try touch-down or other methods for annealing. A gradual cooling from 65ºC to 55ºC or 50ºC can usually make annealing more efficient.e. Annealing temperature needs to be optimized. If necessary, use a temperature gradient to find the optimal annealing temperature. f. Insufficient extension time. Use extension time of two minutes per 1000 base pairs. For DNA fragments hard to be amplified, try 3-4 min per kb. g. Possible problems of PCR thermal cycler. Use a different thermal cycler. h. PCR cycle number is insufficient. Try more PCR cycles. Not to exceed 40 cycles in general.i. The amount of DNA template is too low. Add more DNA templates but within the tolerance limit of the Master Mix.j.Use desalted, PAGE or HPLC-purified PCR primers. k. Use dNTP mix in high quality.l. Add more DNA polymerase appropriately.m. When non-specific DNA fragments are amplified, increase the annealing temperature appropriately.n. Use Positive Control Template and Primers provided to ensure that PCR conditions are optimal.o. Make sure all components necessary for multiplex PCR are added. 2. Presence of non-specific PCR products or DNA smear.a. Increase the annealing temperature by 2-5ºC.b. Adjust PCR cycles.c. Reduce the amount of DNA template.d. Assemble the PCR reactions on ice. Non-specific products are produced easily if PCR reactions are set up at room temperature.e. Use less amount of DNA polymerase, or use Multiplex PCR Kit and PCR Enhancer .f. Decrease the extension time appropriately.

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