UltraBio™ Plant Direct PCR Master Mix

UltraBio™ 植物直接 PCR 预混液

UltraBio™ Plant Direct PCR Master Mix

P751566-100T

100T

2X

-20°C储存,避免反复冻融

超低温冰袋运输

-20℃保存。适当避免反复冻融

阿拉丁生产的UltraBio™ Plant Direct PCR Master Mix (2X)，即UltraBio™植物样品直接PCR预混液(2X)，是一种使用特别便捷的专门用于植物样品的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。本产品含有耐叶绿素的EnzymoPure™ plant DNA聚合酶，这是一种经过基因工程改造的非常高效的适用于植物样品直接PCR检测的耐热DNA聚合酶。对于植物叶片等样品可以直接PCR检测，无须提取DNA。本产品对玉米、丝瓜、甜椒、豆角、黄豆、辣椒、黄瓜、茄子、番茄等植物样品中的叶绿素、多糖、多酚等各种PCR抑制剂表现出超强的抗性，对于新鲜采集的、4℃保存或低温冻存的这些植物的叶片、嫩种子等，都无需进行DNA提取纯化，可以直接用于PCR扩增目的DNA。所需样品少，耐受能力高。本产品用于20μl的PCR扩增体系时，通常仅加入0.1-1mm直径叶片即可顺利完成PCR检测。本产品提供了一对阳性对照引物，便于确认PCR检测效果。该对引物是多种植物的通用引物，可以扩增多数植物叶绿体中高度保守的IRB18基因的297bp DNA片段。本产品用于植物叶片检测的效果参考图1。图中可见，本产品对多种常见植物叶片都有很好的PCR扩增效果。 图1. 阿拉丁生产的UltraBio™ Plant Direct PCR Master Mix (2X)扩增图中所示不同植物叶片中目的基因的检测效果图。使用UltraBio™ Plant Direct PCR Master Mix (2X)及本产品提供的Control primer mix，在PCR体系中直接加入不同植物叶片，用于扩增IRB18基因的297bp DNA片段后的电泳效果图。M, DNA marker )；1, 豆角(Cowpea)；2, 黄豆(Soybean)；3, 丝瓜(Sponge gourd)；4, 甜椒(Capsicum)；5, 玉米(Maize)；6, 黄瓜(Cucumber)；7, 辣椒(Pepper)；8, 茄子(Eggplant)；9, 番茄(Tomato)。本产品扩增不同长度目的基因的效果参考图2。图中可见，本产品以番茄叶片为模板可以直接PCR扩增出长达5kb的DNA片段。 图2. 阿拉丁生产的UltraBio™ Plant Direct PCR Master Mix (2X)直接以番茄叶片为模板扩增不同长度的目的基因的效果图。M, DNA marker ); 1, IRB18(297bp)；2, actin(693bp)；3, rbcL(1201bp)；4, rpoC2(2069bp)；5, rpoC2(3079bp)；6, rpoC2(3942bp)；7, rpoC2(5000bp)。本产品扩增获得的PCR产物带有3'-dA overhangs的粘性末端，可直接用于和T载体连接进行TA克隆。 用途 ： 植物叶片等比较柔嫩的组织样品基因组DNA中目的基因的直接扩增、基因分型(如基因缺失等)，以及基因敲除或转基因植物基因型分析。 失活或抑制 ： 酚氯仿抽提可以使Easy-Load? Plant Direct PCR Master Mix (2X)中含有的PlantTaq? DNA Polymerase失活。本产品如果用于50微升的PCR反应体系，两种包装的本产品分别足够用于40个和200个反应；如果用于20微升的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 ： 由于PCR反应非常灵敏，在使用Easy-Load? Plant Direct PCR Master Mix (2X)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。设置PCR反应体系时，20μl和50μl PCR体系中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，可以直接添加到PCR体系中，无需进行任何额外处理。经测试本产品适用于柔嫩新鲜种子的直接PCR，但不适用于干硬种子的直接PCR。PCR反应结束之后，酌情3000-5000g离心3-5min以沉淀植物组织碎片，便于吸取上清用于电泳分析等。需自备Nuclease-Free Water。推荐选购阿拉丁生产的ST876 ? Ultrapure Water (DNase/RNase-Free, Sterile)。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Easy-Load™ Plant Direct PCR Master Mix (2X)和Control primer mix (10μM each)，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：试剂20μl体系50μl体系终浓度Nuclease-Free Water9μl22.5μl-Easy-Load™ Plant Direct PCR Master Mix (2X)10μl25μl1X引物混合物(10μM each)1μl2.5μl0.5μM each植物样品0.1-1mm直径0.3-3mm直径-总体积20μl50μl-注意：(a)模板使用量：作为PCR模板的植物组织用量，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。(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，使液体和待检测植物组织积聚于管底。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。2.PCR反应参数的设置可以参考如下表格： 步骤循环数温度时间说明1-94℃5min起始变性230-4094℃30sec变性55℃30sec退火68℃2min/kb延伸3-68℃10min最终延伸4-4℃长时间保持临时存放注意：a.起始变性(94℃，5min)可以使植物组织样品裂解，释放出可用于PCR扩增的基因组DNA。b.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，包括温度、时间和循环数等。c.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。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。向PCR体系中尝试加入适合扩增高GC含量DNA片段的试剂。c.目的片段过长。尽管PlantTaq™ DNA Polymerase可以扩增最长达5kb的DNA片段，但大多数时候更适合扩增2-3kb以下的片段。对于过长的目的片段的扩增，需要适当优化引物和PCR反应参数。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，可以在耐血体积范围内适当加大样品用量，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。j.对PCR引物进行脱盐甚至PAGE胶或HPLC纯化。k.当产生较多非特异性条带时，可以适当提高退火温度。l.注意设置适当的阳性对照和阴性对照通常会对于实验结果的判断有很大帮助。2.杂带较多或条带弥散a.退火温度提高2-5℃。b.减少植物组织样品的用量。c.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。d.如果模板的GC含量过高，需要考虑加入适合扩增高GC含量DNA片段的试剂。e.适当缩短延伸时间。 Application ： DNA amplification from plant leaves or other tender tissues; genotyping; genotype analysis of transgenic plants.Inactivation or inhibition: Activities of PlantTaq™ DNA Polymerase contained in this product can be inactivated by phenol-chloroform extraction. Inactivation or inhibition ： Activities of PlantTaq™ DNA Polymerase contained in this product can be inactivated by phenol-chloroform extraction. Precautions ： Because the 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.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 product, but not dry and hard seeds. Instructions for Use ： 1. Prepare PCR reactions:a. Thaw the Easy-Load™ Plant Direct PCR Master Mix (2X) and Control Primer mix (10μM each) at room temperature. Mix well gently by inversion and centrifuge briefly. Keep the reagents on ice.b. Assemble PCR reactions on ice as follows:ComponentVolume (μl)Volume (μl)Final ConcentrationNuclease-Free Water9μl22.5μl-Easy-Load™ Plant Direct PCR Master Mix (2X)10μl25μl1XPCR Primer Mix (10µM each)1μl2.5μl0.5μM eachPlant Samples0.1-1mm diameter0.3-3mm diameter-Total Volume2050-Note 1: 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 product, 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 PCR reactions. One to two small seeds such as tomato seeds can be added directly into PCR reactions for DNA amplification. In a 50μl of PCR reaction, the diameter of the 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 in 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).Note 2: Each Primer at a final concentration of 0.5 μM normally works well, but primer concentration can be optimized between 0.1-1.0 μM. Increase the primer concentration when amplification efficiency is low and decrease the primer concentration when non-specific PCR products are amplified. a. Mix well by gentle vortex or pipetting. Centrifuge briefly to allow liquid to accumulate at the bottom of the PCR tube.b. (Optional) When using a thermocycler without a hot lid, add a drop of mineral oil to the reaction to avoid evaporation. 2. Transfer the PCR reactions to a thermal cycler and run thermocycling conditions as follows:StepTemperatureDurationCyclesInitial Denaturation94℃5 min1Denaturation94℃30 sec30-40Annealing55℃30 secExtension68℃2 min/kbFinal Extension68℃10 min1Holding4℃--Note 1: Denaturation at 94℃for 5 min is to lysis plant tissues to release genomic DNA for PCR amplification.Note 2: Optimize PCR running conditions based on the template, primer sequence, amplicon length or GC content, etc. Note 3: Use 35 cycles for your first try to ensure obtaining the expected DNA fragments.3. Analyze PCR products by agarose gel electrophoresis: After amplification, centrifuge PCR reactions at 3000-5000×g for 3-5 minutes to collect the supernatant for agarose gel electrophoresis. This product contains premixed loading dye that enables reactions to be loaded directly on the agarose gel.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. In this case, GC-Rich buffer suitable for amplifying GC-rich DNA fragments is recommended, and PCR reaction parameters should be adjusted accordingly.c. DNA fragment is too long to be amplified. Although PlantTaq™ DNA polymerase contained in the Master Mix can amplify DNA fragments up to 5 kb in length, it is more suitable for amplification of DNA fragments less than 2-3 kb. Optimize primer sequence or other PCR parameters to amplify longer 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 for each template-primer pair combination. f.Insufficient extension time. Use an 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 plant tissues but within the tolerant limit of the Master Mix, or try nested PCR or secondary PCR. Nested PCR is to design a pair of PCR primers inside the originally designed PCR primers, and then conduct PCR amplification again with the diluted first PCR product as a template. Instead, secondary PCR uses the same primers for PCR amplification again with the diluted first PCR product as a template. Nested PCR can usually remove the non-specific DNA amplification, but secondary PCR can not. j. Use desalted, PAGE or HPLC-grade PCR primers. k. When non-specific DNA fragments are amplified, increase the annealing temperature appropriately.l. Positive control and negative control are always recommended when optimizing PCR reactions. 2. Presence of non-specific PCR products or DNA smear.a. Increase the annealing temperature by 2-5℃.b. Reduce the amount of plant tissues in PCR reactions.c. Assemble the PCR reactions on ice. Non-specific products are produced easily if PCR reactions are set up at room temperature.d. Supplement the PCR reactions with an appropriate amount of DMSO or other reagents that can improve the amplification of GC-rich templates.e. Decrease the extension time appropriately.