AmpliTaq Gold™ 360 DNA Polymerase, 1000 Units - FAQs

当我用凝胶电泳检测时，我的寡核苷酸似乎没有显示出正确长度。这是为什么？

寡核苷酸应该在含有7M尿素的聚丙烯酰胺凝胶上检测，并且应该与50%的甲酰胺溶液一起上样以避免压缩和形成二级结构。长度相同但是组成不同的寡核苷酸可以产生不同的电泳结果。dC的迁移最快，其次是dA、dT，最慢的是dG。带有核糖核酸的寡核苷酸容易产生模糊的电泳条带，并且通常存在二级结构问题。

我所使用的引物刚开始时可以用来做PCR的，但是随着时间的推移它失效了。这是什么情况？

在进行PCR之前应将引物按单次使用的量分装。将分装的引物在94度下加热1分钟。在加入PCR反应体系前快速在冰上冷却引物。有些引物可能会自己退火或者自己蜷缩起来。

在我定制的寡核苷酸管中没有看到沉淀。我是否应该要求换货？

引物的干燥方法会使其附在管壁上形成一个薄层而不是附在底部，因此并不是总能看到沉淀，这种情况下产品仍可以正常使用。

在我的寡核苷酸管子底部有球状的沉淀。这是什么？我的寡核苷酸还能再用吗？

如果寡核苷酸受热过度，它看上去就像一个“球”状沉淀附在管子的底部。这应该不会影响到寡核苷酸的质量，并且该寡核苷酸应该是易溶于水的。

我的冻干寡核苷酸是绿色的。还能再用吗？

如果寡核苷酸看上去是绿色的，最有可能是墨水落入离心管中。该寡核苷酸应仍然是具有功能的。您可通过乙醇沉淀法去除该颜色。

我刚刚收到了我的引物，而它们看上去发黄。还能再用吗？

大多数情况下，颜色不会影响PCR或者其它实验应用，这通常是由于合成过程中使用的碘引起的。然而，也有一些例外情况。引物过度干燥可能引起寡核苷酸发棕色，如果出现这样的情况，引物可能就失效了。

我的引物有一个额外插入的碱基。这是什么情况？

如果脱三苯甲基没有正确出现，并且/或者如果合成仪出现错误并递送错误的碱基，则会在你的引物上出现一个额外插入的碱基。如需帮助，请联系 techsupport@lifetech.com。

在我序列的中间缺失了一个核苷酸。这是什么情况？

生产引物时，可能会在任何一轮的寡核苷酸延伸反应中出现两种可能性：

1.新增的碱基没有正确地脱三苯甲基，少增加了一个碱基，但是却可以进行下一轮反应延伸。
2.三苯甲基被去除了，但在加入下一个碱基之前没有偶联或者正确盖帽，因此寡核苷酸链可继续延伸。

我订购了引物，在寡核苷酸3’末端和5’末端引入酶切位点，且进行了脱盐纯化。当我试图对PCR产物进行亚克隆时，得到了细胞集落非常少。我检查了所有条件，似乎只有寡核苷酸可能存在问题。您能为我解释一下为什么发生这样的情况吗？

为了实现全长寡核苷酸序列，建议您使用纯化水平更高的寡核苷酸。增加限制性内切酶位点，会使20-25-mer的寡核苷酸上增加10个或者更多的碱基，使引物长度超过30个的碱基，同时使脱盐后全长序列的百分比会相对较低。此外，引物合成寡核苷酸时从3’末端合成到5’末端，序列的5’末端会出现失败序列。因此，在引物5’末端引入的限制性内切酶位点可能因为效果不佳，导致碱基缺失。

我的寡核苷酸在重新溶解后得率低。这是什么情况？

该寡核苷酸可能没有完全溶解。在加入TE缓冲液后，确保寡核苷酸震荡混匀30秒，并且/或者用移液器吹打10次以上。引物可能会粘在离心管的壁上，所以在重悬寡核苷酸时，也应该“冲洗”离心管壁。

我收到了我的引物订单，但其中的产率比我订购的规格要低。为什么会这样？

定制规格指的是开始合成的规格，或者用来合成您的寡核苷酸的起始材料量。根据纯化水平和效率，你将获得比原始合成规格更少的得率。然而，根据起始合成的规模，我们确实有一个最低得率保证，你可以 点击此处查看。.

我在孔中看到可被溴化乙锭染色（EB）的高分子量物质残留。为什么会出现这样的情况？

循环次数太多或者在反应体系中加入太多DNA时，会出现这种残留物。可以试试在上样之前把样品加热到65度，然后再放到冰上。

我的PCR反应得到了不想要的产物。是什么造成的？您有什么好的建议吗？

这种情况可能存在的原因和我们提供的建议如下：

•引物设计：可试试更长的引物以避免结合到其它位点，避免3’端存在3个连续的G或C核苷酸。
•退火温度：可增加退火温度以增加特异性。
•镁离子浓度：试试降低镁离子浓度。
•DNA污染：可使用防气溶胶的枪头并隔离工作区域以避免污染，可使用UNG/UDG技术来防止前次PCR产物遗留。

我的PCR产物中没有条带。可能的原因是什么？

以下一些原因可能造成您的PCR实验失败：

•存在50mM的NaCl，抑制了酶的活性。
•反应体系有太多KCl。KCl含量不应超过50mM。
•使用的退火温度有误。
•变性不完全（时间必须足够长，温度必须足够高）。
•模板有一长串的GC序列[Woodford et al. (1995) Nucleic Acids Res 23:539声称，从扩增反应中去除所有的钾成分，模板中的富GC区域就足以解链，从而达到可进行PCR的程度]
•存在10%的DMSO，部分抑制了Taq聚合酶活性。
•（血液样本）存在氯高铁血红素，抑制了Taq聚合酶活性。
•使用超辐射（>2500mJ/cm2的强度处理）矿物油可能抑制或者降低PCR产物的得率[Dohner (1995) Biotechniques 18:964]。
•在PCR之前不要使用木质牙签挑取克隆或从胶中挖出DNA。有报道表明这种做法会抑制PCR反应[Lee （1995）Bio Techniques 18:225]。
•存在其它Taq DNA聚合酶抑制剂（如靛蓝染料、亚铁血红素、黑色素等）。可向PCR体系添加牛血清白蛋白（BSA）（约160-600μg/mL）、增加Taq酶的总量、和/或者通过增加PCR的体积来稀释抑制剂。

我希望得到的片段最终产率很低。我哪里做得不对？如何可以提高我的得率？

请查看我们关于如何增加得率的以下几点建议：

•在PCR之前不要使用木质牙签挑取克隆或从胶上挖出DNA。有报道表明这种做法会抑制PCR反应[Lee （1995）Bio Techniques 18:225]。
•没有使用足够的酶。
•变性/延伸温度太高，酶过早失活。
•DMSO过量（>10%）。
•退火温度有误：从低于计算的Tm值5度的温度开始，用不同的退火温度来进行一系列反应。
•循环次数太少。
•Mg2+不足或过多。
•引物设计不佳：再次确认引物序列与模板序列，引物对应该有相似的解链温度，避免引物3’末端有互补序列。
•遗留抑制剂（如血液、血清）。
•变性时间过短。基因组DNA和病毒DNA可能需要10分钟的变性时间。
•相对于所扩增产物的大小，延伸时间可能不足。
•使用超辐射（>2500mj/cm2的强度处理）矿物油可能抑制或者降低PCR产物的得率[Dohner (1995) Biotechniques 18:964]。
•模板有一长串的CG序列[Woodford et al. (1995) Nucleic Acids Res 23:539声称，从扩增反应中去除所有的钾成分，模板中的富GC区域就足以解链，从而达到可进行PCR的程度]。此外， 1.0M三甲基甘氨酸与6%-8%的DMSO或5%的DMSO与1.2-1.8M三甲基甘氨酸的组合，均可用于扩增富GC模板[Baskaran (1996) Genome Res 6:633]。
•若存在其它Taq DNA聚合酶抑制剂（如靛蓝染料、亚铁血红素、黑色素等），可以向PCR体系添加牛血清白蛋白（BSA）（约160-600μg/mL）、增加Taq酶的总量、和/或者通过增加PCR的体积来稀释抑制剂。加入的BSA浓度可能取决于存在的抑制剂含量及类型。此外，无脂肪酸、乙醇沉淀的BSA或Fraction V BSA应该全部都有效。

我在PCR后看见拖尾效应。这是什么原因造成的？

出现拖尾效应时，可以确认有无以下几点原因：

•酶、引物、镁离子浓度以及/或者dNTP浓度过高。
•退火温度相对所用的引物来说过低。
•循环次数太多。
•退火和延伸的时间太长。
•引物设计不佳或存放时间过久。
•最初使用的模板太多，可试试以104-106个拷贝的模板量起始。
•可以考虑使用添加剂或者 PCR Optimizer试剂盒（货号K122001），尤其在当您使用Taq酶扩增时，您觉得引物应该有效，或者之前用过有效的前提下特别推荐进行这样优化。

如果我选择混合碱基的方法（如GC）来生产我的寡核苷酸，是应该按照50/50的比例混合吗？

不，我们不保证有50/50的混合碱基。例如如果需要混合GC碱基，我们的合成仪会使用正常数量一半的G碱基和正常量一半的C碱基。这一计算并未将偶联效率考虑在内。因此，可能会出现30/70的碱基混合物。

这些寡核苷酸的质量是如何控制的？

对于25、50和 200 nmol的脱盐和柱体纯化的DNA寡核苷酸，100% 通过A260 分析进行质控。此外随机选取25%的寡核苷酸产物样品进行毛细管电泳或质谱检测。对于用户订购的25和50 nmol规格的脱盐DNA寡核苷酸，其分析过程中还100%执行实时数字三苯甲基监控。对于用户订购的1和10 μmols规格脱盐DNA寡核苷酸，全部规格的HPLC和PAGE纯化的DNA寡核苷酸，大多数含有3’端和/或5’端修饰的DNA寡核苷酸，以及RNA寡核苷酸都会100%执行 A260分析、毛细管电泳或质谱检测。

对于96孔板或384孔板的订单来说，我需要订购多少条寡核苷酸？

在整个平板形式的订单中，96孔板每板平均不少于24条寡核苷酸；384孔板每板平均不少于192条寡核苷酸。

为什么不同的程序会计算出不同的Tm值？

Tm值并不是绝对的——它们是实际存在的熔解温度范围的一个近似值。对于一条给定的寡核苷酸，熔解温度不仅与盐的含量有关，也与碱基组成和在反应液中不能精确定量的引物浓度有关，熔解温度范围会存在10-15度的波动。您不应仅仅使用某个给定Tm值进行实验。Tm是指在该温度时，有50%的引物与其互补序列以双链DNA分子的形式存在。Tm是确定PCR退火温度的必要条件。合理的退火温度范围介于55°C到70°C之间。退火温度通常比引物的Tm低5°C。因为绝大多数公式提供的是预估的Tm值，所以这里的退火温度只是作为一个起始温度进行尝试。在逐渐增加退火温度的条件下进行多次反应并分析，可以增加PCR的特异性。

为什么不能针对较大的寡核苷酸提供HPLC或柱体纯化？

因为随着寡核苷酸长度增加，在从正确的产物中分离出合成失败的寡核苷酸时，柱体纯化方法的效率会越来越低。在这种情况下，PAGE纯化是相对理想的选择。

何为Value Oligos？

Value Oligos是最经济、快速的寡核苷酸订购方法。其可以选择5-40mer、25或50纳摩尔规格，同时包含了一系列可满足您需求的纯化选项，且可实现次日发货（仅适用于美国客户）。其订购费用按照每条寡核苷酸而不是每个碱基来计算。Value Oligos不提供修饰选项。Value Oligos与我们的标准寡核苷酸采用相同的生产流程，有着相同的质控标准。

我该如何计算自己引物的熔解温度？

计算引物Tm的常用公式如下：

Tm （°C） = 2 (A+ T) + 4 (G + C)

Thermo Fisher Scientific可以为我的引物提供何种类型的修饰？

请查看此列表（https://www.thermofisher.com/cn/zh/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/oligo-ordering-details/oligo-modification-options.html）了解我们所提供的标准修饰选择。如果您未能找到自己想要的修饰选项，请发送邮件到LifeScience-CNTS@thermofisher.com 联系我们的技术支持团队，询问我们是否能够满足您的要求。

我该如何测定全长寡核苷酸的百分比？

全长寡核苷酸的百分比取决于化学合成的偶联效率。此类化学合成的平均偶联效率接近99%。计算全长寡核苷酸的百分比时，可采用以下公式：0.99n-1。因此，离心管里79%的寡核苷酸分子长度是25碱基，其余的寡核苷酸长度则<25个碱基。如果您关心如何着手制备全长核苷酸，可以考虑采用柱体纯化、PAGE或HPLC纯化。

偶联效率为何很重要？

在DNA合成过程中，偶联效率是一个重要因素，这是因为其影响具有累积效应。下表显示了1%的偶联效率差异所引发的效应，以及在不同长度寡核苷酸合成过程中，该种差异对全长产物的得率造成的影响。即使是合成相对较短的20个碱基的寡核苷酸，哪怕偶联效率存在1%的差异，也会造成全长产物得率相差15%以上。

添加的碱基数量，99%偶联效率下的全长产物的百分比，未能合成全长产物的百分比，98%偶联效率下的全长产物的百分比，未能合成全长产物的百分比：

1， 99， 1， 98， 2
2， 98.01， 1.99， 96.04， 2.96
3， 97.03， 2.97， 94.12， 5.88
10， 90.44， 9.56， 81.71， 18.29
20， 81.79， 18.21， 66.76， 33.24
30， 73.79， 26.03， 54.55， 63.58
50， 60.5， 39.5， 36.42， 63.58
95， 38.49， 61.51， 14.67， 85.33

你们供应的寡核苷酸可以保证的最低得率是多少？

合成的规模是合成的起始用量，而不是担保的终产量。我们以最小OD单位保证寡核苷酸的总得率。您可 点击此链接(https://www.thermofisher.com/cn/zh/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/oligo-ordering-details/oligo-minimum-yield-guarantee.html)查看我们提供寡核苷酸时所担保的最低得率。

你们能提供一些有助于设计引物的资源吗？

有的。OligoPerfect Designer即可为测序、克隆或检测等应用设计引物。

关于如何设计自己的PCR引物，您能为我提供一些指导原则吗？

以下指导原则可能有助于您设计自己的PCR引物：

•通常来说，引物长度以18-30个核苷酸为佳。
•可尽量使引物的熔解温度（Tm）处在65°C到75°C之间，同时确保引物间的熔解温度差值在5°C之内。
•如果您的引物的Tm非常低，可尽量找一段高GC含量的序列，或者稍微延长一下引物的长度。
•尽量保证GC含量在40%到60%之间，同时确保引物的3’末端为C或G，从而促进结合效率。
•通常情况下，可在引物的限制性内切酶位点的5’末端增加3-4个核苷酸，促进有效地酶切。
•尽量避免出现二级结构区域，同时确保富含GC和富含AT的区域分布均衡。
•尽量避免连续出现4个及以上的相同碱基，或者某两个核苷酸连续重复出现（例如，ACCCC或ATATATAT）。
•避免引物内同源性（引物内有超过3个碱基互补）或引物间同源（正反向引物间有互补序列）。这些情况会引起自身二聚体或引物二聚体，而不是退火到所需的DNA序列上。
•如果您使用引物进行克隆，建议纯化水平至少达到柱体纯化的水平。
•如果您采用引物进行突变研究，请尽量将错配碱基放在引物序列的中间位置。
•如果您使用引物进行PCR反应，然后再进行TOPO克隆，则引物不应该有磷酸化修饰。

如需了解更多关于引物设计的提示和工具，请点击此处。

双温度实验方案（两步法）如何操作？您建议在什么时候使用？

退火温度相对较高时，您可以选择使用双温度实验方案。此时，退火和延伸步骤放在一起进行，例如，两者都可在>62°C的温度下发生。相对常规的三温度实验方案（三步法），双温度实验方案的优势是速度快得多。

怎样才能促进含发夹结构或者高GC含量的模板扩增？

您可以尝试加入5%-10%的DMSO、至多10%的甘油或者1-2%的甲酰胺，或者联用这些方法促进这些难扩增模板的扩增。注意：使用助溶剂会降低您的引物的最佳退火温度。

为什么高GC含量的模板很难扩增？

高GC含量的模板通常具有较高的熔解温度，在正常的反应条件下可能不能完全变性。

热启动PCR是什么意思？

热启动是一种可防止DNA在不恰当的时机出现扩增的方法。为此，您可在冰上建立PCR反应，从而防止DNA聚合酶一开始便处于活性状态。更简单的做法则是使用一种“热启动”酶——这种酶在供应时为非活性状态，高温加热步骤之后才会活化。

PCR的主要步骤有哪些？

PCR的主要步骤是：变性、退火、延伸。通常情况下，需要将模板加热到较高温度（大约94-95°C）使双链DNA变性成为单链。然后，将温度降到50-65°C，使引物退火到模板上互补的碱基配对区域。之后，再将温度升高到72°C，使聚合酶结合到模板并合成一条新DNA链。

AmpliTaq Gold 和 Platinum Taq之间有何区别？

AmpliTaq Gold和Platinum Taq都是热启动酶，均支持在无冰的情况下在实验台上配制反应体系。AmpliTaq Gold是一种化学修饰的热启动酶，以非活性状态提供。在95°C下，该酶可在10分钟后完全热激活。Platinum Taq是一种抗体介导的热启动酶。抗Taq抗体会与酶结合并抑制酶的活性，直至PCR反应的热变性阶段（30秒到2分钟），该酶的活性才被激活。

您能对比一下你们提供的DNA聚合酶在保真度、最大扩增长度及3′端是否添加A尾等方面的差别吗？

请见下面的比较表： 酶，相对保真度，扩增长度，3'端A 尾 Taq， 1， <5 kb， + Platinum Taq， 1， <10 kb， + AccuPrime Taq， 2，<5 kb， + Platinum Pfx， 26， <12 kb， - AccuPrime Pfx， 26， <12 kb， - Pfx50， 50， <4 kb， - Platinum Taq HiFi， 6， <20 kb， +/- AccuPrime Taq HiFi， 9， <20 kb， +/- AmpliTaq， 1， <5 kb， + AmpliTaq Gold， 1， <5 kb， + AmpliTaq Gold 360， 1，<5 kb， + Taq酶错配率：1 x 10-4至2 x 10-5碱基/每次复制

My oligonucleotide does not appear to be the right length when I checked by gel electrophoresis. Why is this?

Oligos should be run on a polyacrylamide gel containing 7 M urea and loaded with a 50% formamide solution to avoid compressions and secondary structures. Oligos of the same length and different compositions can electrophorese differently. dC's migrate fastest, followed by dA's, dT's, and then dG's. Oligos containing N's tend to run as a blurry band and generally have a problem with secondary structure.

The primers I am using worked for PCR initially, but over time, have stopped working. What happened?

Primers should be aliquoted for single use before PCR set-up. Heat just the aliquoted primers to 94 degrees for 1 min. Quick chill the primer on ice before adding to the PCR reaction. Some primers may anneal to themselves or curl up on themselves.

I don't see a pellet in my oligo tube order. Should I ask for a replacement?

The drying method dries the primer in a thin layer along the sidewalls of the tube instead of the bottom, therefore a pellet is not always visible and should still be ready to use.

There is a ball-shaped pellet at the bottom of my oligo tube. What is this and can I still use my oligo?

If the oligo was overheated, it will appear as a “ball”-shaped pellet attached to the bottom of the tube. This should not affect the quality of the oligo, and the oligo should be readily soluble in water.

There is a green color in my lyophilized oligo. Can I still use it?

If an oligo appears green in color, this is most likely due to ink falling into the tube. The oligo should still be fully functional. The color can be removed by doing an ethanol precipitation.

I just received my primers and they look yellow. Can I still use them?

Most of the time the color should not affect PCR or any other experimental application since typically it is caused by the iodine used in the synthesis. There are some exceptions, however. Brown oligos can also be caused by the primer being overdried, and if this is the case, the primer may not work.

My primer has an extra inserted base. How could this happen?

If detritylation occurs inappropriately and/or if the synthesizer has an error and delivers the wrong base, an extra inserted base can occur in your primer. Please contact techsupport@thermofisher.com for assistance.

I'm missing a nucleotide in the middle of my sequence. How could this happen?

There are two possibilities that could occur in any round of extension when creating your primer:

1.The added base is not detritylated correctly, missing one base addition but allowing possible extension in the next round.
2.The trityl group was removed, but not coupled or capped correctly before addition of the next base, allowing the chain to continue.

I ordered a primer with restriction enzyme sites flanking the 3' and 5' ends of my oligo with desalted purification. When trying to subclone the PCR product, I get very few colonies. I have tested all conditions, and it seems to be the oligo causing the problem. Can you explain why this happened?

Better purification of the oligos is recommended to provide you with full-length oligo sequence. Adding restriction sites adds on 10 or more bases to the basic 20-25-mer, making primers longer than 30 bases with a relatively low percentage of full-length sequences after desalting. Additionally, failure sequences occur at the 5' end of the sequence as oligos are generated from 3' to 5' end. Therefore, restriction sites introduced at the 5' end of primers can be compromised, resulting in missing bases.

I'm getting low yield of my oligo upon reconstitution. What happened?

The oligo may not have been fully solubilized. After addition of TE buffer, make sure the oligo was vortexed for a full 30 seconds and/or pipette up and down more than 10 times. Primers may be present along the sides of the tubs, so when resuspending the oligo, the sides of the tubes should be “rinsed” too.

I received my primer order, but the yield is lower than the scale that I ordered. Why is this?

The scale that is ordered refers to the starting synthesis scale, or amount of starting material used to create your oligo. Based on purification and efficiency, you will receive less than the starting synthesis scale. However, we do have a minimum yield guarantee based on the starting synthesis scale which can be found here: https://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/oligo-ordering-details/oligo-minimum-yield-guarantee.html.

I'm seeing high molecular weight EtBr stainable material left in wells. Why is this happening?

This artifact occurs when either too many cycles were performed or too much DNA is added to the reaction. Try heating to 65 degrees C and putting sample on ice before loading.

I'm getting an unexpected product when performing PCR. What could be the cause of this and what do you suggest I try?

Please see the following possibilities and suggestions we have:

-Primer design: try longer primers to avoid binding at alternative sites, avoid 3 consecutive G or C nucleotides at the 3' end.
-Annealing temperature: increase annealing temperature to increase specificity.
-Mg2+ concentration: try a lower concentration.
-DNA contamination: use aerosol tips and separate work area to avoid contamination, use UNG/UDG technique to prevent carryover.

I'm getting no bands from my PCR product. What could cause this?

Here are some reasons why your PCR experiment may be failing:

-NaCl at 50 mM will inhibit the enzyme.
-Too much KCl in the reaction. Do not exceed 50 mM.
-Incorrect annealing temperature was used.
-Incomplete denaturation (time and temperature must be long and high enough).
-Template had long runs of GC's [Woodford et al. (1995) Nucleic Acids Res 23:539 show that by eliminating all potassium from the amplification reactions, GC-rich regions in templates are sufficiently destabilized to allow PCR].
-10% DMSO partially inhibits Taq.
-Hemin (in blood samples) inhibits Taq.
-Use of super-irradiated (treated with >2500 mJ/cm2) mineral oil will either inhibit or decrease yield of PCR product [Dohner (1995) Biotechniques 18:964].
-Do not use a wooden toothpick to pick colonies or scoop out DNA from a gel prior to PCR. It has been reported that this technique can inhibit PCR [Lee (1995) BioTechniques 18:225].
-Other inhibitors of Taq DNA polymerase were present (e.g., indigo dyes, heme). Add BSA to the PCR, increase the amount of Taq, and/or increase the volume of the PCR to dilute out them inhibitor.

I'm getting low yield of my desired fragment. What am I doing wrong and how can I increase my yield?

Please see our suggestions below to increase yield:

-Do not use a wooden toothpick to pick colonies or scoop out DNA from a gel prior to PCR. It has been reported that this technique can inhibit PCR. [Lee (1995) BioTechniques 18:225].
-Not enough enzyme was used.
-Denaturation/extension temperature was too high and enzyme died prematurely.
-Too much DMSO (>10%).
-Incorrect annealing temperature: run a series of reactions using different annealing temperatures, starting 5 degrees below the calculated Tm.
-Too few cycles.
-Insufficient or too much Mg2+.
-Poorly designed primers: double check primer sequence against template sequence, primers should have similar melting temperatures, avoid complementary sequences at the 3' end of primers.
-Carryover inhibitors (e.g., blood, serum).
-Denaturation time was too short. Genomic and viral DNA can require denaturation times of 10 minutes.
-Not a long enough extension time was used depending on the size of product being amplified.
-Use of super-irradiated (treated with >2500 mj/cm2) mineral oil will either inhibit or decrease yield of PCR product [Dohner (1995) Biotechniques 18:964].
-Template had long runs of GC's [Woodford et al. (1995) Nucleic Acids Res 23:539 show that by eliminating all potassium from the amplification reactions, GC-rich regions in templates are sufficiently destabilized to allow PCR]. Alternatively, a combination of 1.0 M betaine with 6-8% DMSO or 5% DMSO with 1.2-1.8 M betaine can be used to amplify GC-rich templates [Baskaran (1996) Genome Res 6:633].
-Other inhibitors of Taq DNA polymerase were present (e.g., indigo dyes, heme, melanin, etc.). Add BSA to the PCR (~160-600 µg/mL), increase the amount of Taq, and/or increase the volume of the PCR to dilute out the inhibitor. The concentration of BSA to add may be dependent on the amount and type of inhibitor present. Additionally, fatty acid-free, alcohol-precipitated BSA, or Fraction V BSA all should be effective.

I'm seeing smearing after PCR. What is causing this?

Please see some reasons below for seeing smearing:

-The enzyme, primer, Mg2+, and/or dNTP concentration was too high.
-The annealing temperature was too low for the primers being used.
-Too many cycles were used.
-The annealing and extension times were too long.
-Bad or old primers.
-Too much template was used initially, try to start with 104-106 molecules
-Consider using additives or PCR Optimizer Kit (Cat. No. K122001), especially if you feel strongly that the primers should work/have worked before and are using Taq.

If I choose mixed bases, e.g., GC, for my oligo manufacturing, will it be a 50/50 mix?

No, we do not guarantee 50/50 of mixed bases. If a mix of GC bases is requested, for example, the synthesizer would deliver half the normal amount of G and half the normal amount of C. Coupling efficiency is not taken into account. Therefore, it is possible that a mix, such as 30/70, will be delivered.

How are these oligos quality controlled?

For 25, 50, and 200 nmol desalted and cartridge-purified DNA oligos, there is 100% A260 analysis. Random samples of 25% of the oligos produced are tested by either capillary electrophoresis or mass spectrometry. DNA oligos that are desalted and ordered at 25 and 50 nmol scales also have 100% real-time digital trityl monitoring during analysis. Desalted DNA oligos ordered at 1 and 10 µmols, DNA oligos at any scale that are purified by HPLC and PAGE, the majority of the DNA oligos with 3' and/or 5' modifications, and RNA oligos have 100% A260 analysis and capillary electrophoresis or mass spectrometry.

How many oligos do I need to order for a 96-well plate order or a 384-well plate order?

The plate orders must contain an average of 24 or more oligos per plate for 96-well plates or 192 or more oligos per plate for 384-well plates across the entire order.

Why do different programs calculate different Tm values?

Tm values are not absolute - they are an approximation of the melting temperature range which exists. A thermal profile for a given oligo shows a 10-15 degree range of melting depending on the amount of salt but also on the base composition and concentration of primer in the reaction which are not precisely defined. One should not rely solely on the given Tm value as the only one that will work. Tm is the temperature at which 50% of the primer and its complementary sequence are present in a duplex DNA molecule. The Tm is necessary to establish an annealing temperature for PCR. Reasonable annealing temperatures range from 55 degrees C to 70 degrees C. Annealing temperatures are generally about 5 degrees C below the Tm of the primers. Since most formulas provide an estimated Tm value, the annealing temperature is only a starting point. Specificity for PCR can be increased by analyzing several reactions with increasingly higher annealing temperatures.

Why are HPLC or cartridge purification not offered for larger oligos?

As oligos increase in length, the column purification is less effective in separating the failure oligos from the correct products. PAGE purification would be the method of choice in this case.

What are Value Oligos?

Value Oligos are the most cost-effective and fastest way to order oligos. They are available for 5-40-mers, at a 25 or 50 nanomole scale, with a range of purification options to suit your needs, and are eligible for next-day delivery. The cost is calculated per oligo as opposed to per base. Value Oligos are not available with modifications. Value Oligos undergo the same QC standards as our standard oligos with the same manufacturing process.

How do I calculate the melting temperature of my primers?

A common equation used to calculate primer Tm is as follows: Tm (in degrees C) = 2 (A+ T) + 4 (G + C)

What type of modifications does Life Technologies offer for my primers?

Please take a look at this list (https://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/oligo-ordering-details/oligo-modification-options.html) of standard modification options that we offer. If you do not see the modification option you would like, please email our Technical Support team at techsupport@thermofisher.com to see if we can accommodate your request.

How do I determine the percentage of full-length oligonucleotide?

The percentage of full-length oligonucleotide depends on the coupling efficiency of the chemical synthesis. The average efficiency is close to 99%. To calculate the percentage of full-length oligonucleotide, use the formula: 0.99n-1. Therefore, 79% of the oligonucleotide molecules in the tube are 25-bases long; the rest are <25 bases. If you are concerned about starting with a preparation of oligonucleotide that is full-length you may want to consider cartridge, PAGE, or HPLC purification.

Why is coupling efficiency important?

Coupling efficiency is important as the effects are cumulative during DNA synthesis. The numbers below shows the effect of a 1% difference in coupling efficiency and how this influences the amount of full-length product available following synthesis of different length oligos. Even with a relatively short oligo of 20 bases, a 1% difference in coupling efficiency can mean 15% more of the DNA present following synthesis is full-length product.

Number of bases added, 99% coupling full-length, Failures, 98% coupling full-length, Failures:
- 1, 99, 1, 98, 2
- 2, 98.01, 1.99, 96.04, 2.96
- 3,97.03, 2.97, 94.12, 5.88
- 10, 90.44, 9.56, 81.71, 18.29
- 20, 81.79, 18.21, 66.76, 33.24
- 30, 73.79, 26.03, 54.55, 63.58
- 50, 60.5, 39.5, 36.42, 63.58
- 95, 38.49, 61.51, 14.67, 85.33

What are the minimum yield guarantees you offer for your oligos?

The scale of synthesis is the starting point for synthesis, not the guaranteed final amount. We guarantee the total yield of oligonucleotide as a minimum number of OD units. Use this link (https://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/oligo-ordering-details/oligo-minimum-yield-guarantee.html) for the minimum yield guarantees we offer for our oligos.

Do you have any resources to help design primers?

Yes. OligoPerfect Designer can be used to design primers for sequencing, cloning, or detection.

Can you suggest some guidelines that will help me design my PCR primers?

These guidelines may be useful as you design your PCR primers:

- In general, a length of 18-30 nucleotides for primers is good.
- Try to make the melting temperature (Tm) of the primers between 65 degrees C and 75 degrees C, and within 5 degrees C of each other.
- If the Tm of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little.
- Aim for the GC content to be between 40 and 60%, with the 3' of a primer ending in C or G to promote binding.
- Typically, 3 to 4 nucleotides are added 5' of the restriction enzyme site in the primer to allow for efficient cutting.
- Try to avoid regions of secondary structure, and have a balanced distribution of GC-rich and AT-rich domains.
- Try to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT).
- Avoid intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences). These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences.
- If you are using the primers for cloning, we recommend cartridge purification as a minimum level of purification.
- If you are using the primers for mutagenesis, try to have the mismatched bases towards the middle of the primer.
- If you are using the primers for a PCR reaction to be used in TOPO cloning, the primers should not have a phosphate modification.
Read more about primer design tips and tools at https://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/primer-design-tools.html.

How does a two-temperature protocol work and when would you suggest using one?

You may choose to do a two-temperature protocol when the annealing temperature is relatively high. In this case, you would combine the annealing and the elongation steps, i.e., both can occur together at a temperature >62 degrees C. The advantage of a two-temperature protocol is that it is considerably quicker in comparison to the conventional three-temperature protocol.

How can I facilitate the amplification of templates with hairpin-loop structures or high GC-content?

You can try adding 5-10% DMSO, up to 10% glycerol, or 1-2% formamide or a combination of these to facilitate difficult templates. Note: the use of cosolvents will lower the optimal annealing temperatures of your primers.

Why is it difficult to amplify a GC-rich template?

A GC-rich template often has a higher melting temperature and may not denature completely under the normal reaction conditions.

What does hot start PCR mean?

Hot start is a way to prevent DNA amplification from occurring before you want it to. One way to do this is to set up the PCR reaction on ice, which prevents the DNA polymerase from being active. An easier method is a use a ‘hot-start' enzyme, in which the DNA polymerase is provided in an inactive state until it undergoes a high-heat step.

What are the main steps in PCR?

The main steps are: denaturation, annealing, and extension. The template is typically heated to a high temperature (around 94-95 degrees C) allowing for the double-stranded DNA to denature into single strands. Next, the temperature is lowered to 50-65 degrees C, allowing primers to anneal to their complementary base-pair regions. The temperature is then increased to 72 degrees C, allowing for the polymerase to bind and synthesize a new strand of DNA.

What is the difference between AmpliTaq Gold polymerase and Platinum Taq polymerase?

Both AmpliTaq Gold and Platinum Taq are hot-start enzymes that allow you to set up your reactions on the benchtop without the need for ice. AmpliTaq Gold is a chemically-modified hot-start enzyme, provided in an inactive state. Heat activates the enzyme, with full activity after 10 min at 95 degrees C. Platinum Taq is an antibody-mediated hot-start enzyme. The anti-Taq antibodies bind and inactivate the enzyme, until the heat denaturation step of the PCR reaction (30 sec to 2 min), which activates the enzyme.

Can you compare the DNA polymerases you offer by fidelity, maximum amplicon length, and 3' A-overhang?

Please see the comparison below on the following criteria:
Enzyme, Relative Fidelity, Amplicon Length, and 3' Overhang (+/-)
Taq, 1, <5 kb, +
Platinum II Taq Hot-Start, 1, <5 kb, +
Platinum Taq, 1, <5 kb, +
AccuPrime Taq, 2, <5 kb, +
Platinum Taq HiFi, 6, <20 kb, +/-
AccuPrime Taq HiFi, 9 <20 kb, +/-
Platinum Pfx, 26, <12 kb, -
AccuPrime Pfx, 26, <12 kb, -
Pfx50, 50, <4 kb, -
AmpliTaq, 1, <5 kb, +
AmpliTaq Gold, 1, <5 kb, +
AmpliTaq Gold 360, 1, <5 kb, +

Taq error rate: 1 x 10-4 to 2 x 10-5 base/duplication

When using AmpliTaq Gold DNA polymerase, under what conditions would one choose a two-temperature vs. three-temperature PCR?

A two-temperature PCR is commonly used when the primer annealing temperatures are above 60 degrees C. Use a three-temperature PCR when the templates have high G+C content and/or secondary structure, or desired primer annealing temperatures are below 60 degrees C. Please consult the Product Insert for more information on the use of AmpliTaq Gold DNA polymerase.

Is there anything to prevent AmpliTaq Gold DNA polymerase from extending from the 3’ end of a TaqMan probe in a 5’ nuclease assay?

Yes. There is a phosphate group on the 3' end of all TaqMan probes that prevents such extension.

How does AmpliTaq Gold DNA Polymerase differ from AmpliTaq DNA Polymerase?

AmpliTaq Gold DNA Polymerase is a modified form of AmpliTaq DNA Polymerase that contains a proprietary chemical (or so-called hot start molecule) bound to the enzyme's active site. In order to activate the AmpliTaq Gold DNA Polymerase fully, we recommend an initial activation step of 95 degrees C for 10 min when using GeneAmp 10X PCR Buffer I and/or GeneAmp 10X PCR Buffer II and Mg in one of our thermal cyclers. When using GeneAmp 10X PCR Gold Buffer, activation time can be reduced to 5 minutes. Once activation is complete, you can proceed with your standard PCR cycling program (denaturing, annealing, extension, etc).

Does AmpliTaq Gold DNA Polymerase contain exonuclease (proofreading) activity?

No, AmpliTaq Gold DNA polymerase does not contain proofreading activity, however fidelity in PCR amplifications utilizing this enzyme may be improved. High fidelity can be achieved by: 1. Decreasing the final concentration of each nucleotide to 40-50 uM. 2. Using the lowest MgCl2 concentration possible. 3. Using less enzyme. 4. Decreasing extension times. 5. Using the highest annealing temperature possible. 6. Using as few cycles as possible.

Does the fidelity of AmpliTaq DNA Polymerase change in the presence of base analogs?

The fidelity of this PCR enzyme is affected in two ways. First, AmpliTaq DNA Polymerase typically binds to and incorporates base analogs less efficiently than conventional dNTPs, which means that polymerase activity is lower in reactions that contain base analogs. Second, the analog may pair with more than one conventional complementary template base, so the analog may be incorporated at an increased level compared to conventional dNTPs. For the best fidelity, we recommend that base analogs are included at low concentrations in the reaction.

What is the expected half life of AmpliTaq DNA Polymerase at 95 degrees C?

The half-life of AmpliTaq DNA Polymerase at 95 degrees C is 40 min. During PCR, the sample is only incubated at the programmed temperature for approximately 20 seconds. Therefore, the cycling half-life of AmpliTaq Gold at 95 degrees C is approximately 100 cycles.

Example: AmpliTaq DNA Polymerase experiences about 20 seconds at 95 degrees C per PCR cycle. The t_1/2 is at least 33 minutes; (35-40 min). Therefore, 33 min/20 sec/cycle = 100 cycles. 100 PCR cycles reduces enzyme activity by 50%.

What is "Hot-start" PCR?

Hot-start is a technique commonly used to improve the sensitivity and specificity of PCR amplifications. The major obstacle to obtaining highly sensitive and specific amplifications appears to be competing side reactions such as the amplification of non-target sequences (mis-priming) and primer oligomerization. In an otherwise optimized PCR amplification, most non-specific products can be attributed to pre-PCR mispriming. Mispriming can occur any time all components necessary for amplification are present at permissive temperatures (below optimal annealing temperature) such as during reaction set up. A hot start can be performed either manually or can be automated utilizing AmpliTaq Gold DNA Polymerase.

In the manual hot-start technique a key component necessary for amplification, such as the enzyme, is withheld from the reaction mix until the reaction reaches a temperature above the optimal annealing temperature of the primers. Once this temperature is reached, the missing component is added and the PCR amplification is allowed to proceed. Because a key component was withheld from the reaction at permissive temperatures, competing side reactions are minimized and specific amplification occurs.

AmpliTaq Gold DNA Polymerase facilitates the automation of the hot start technique and decreases the potential for contamination. AmpliTaq Gold DNA Polymerase is a modified form of AmpliTaq DNA Polymerase. Once activated, AmpliTaq Gold DNA Polymerase performs just as AmpliTaq DNA Polymerase does. Since it is provided in its inactive form, it can be added to a reaction without the fear of pre-PCR misprimed primers being extended. Once all of the components for amplification have been added to a tube, the reaction is heated to 95C for 5 - 10 minutes. This incubation activates the enzyme and allows the reaction to proceed normally.

How much MgCl2 should be added to the PCR amplification when using AmpliTaq DNA Polymerase or AmpliTaq Gold DNA Polymerase?

The standard starting point is a final concentration of 1.5 mM magnesium ion. Since each molecule of dNTP (total 0.8 mM per reaction at 200 µM each) binds a magnesium ion, 0.8 mM magnesium ions are unavailable for AmpliTaq DNA Polymerase to use; hence, 0.7 mM free magnesium ions will be available as a cofactor for Taq's polymerization activity. It is important to note that there are other substrates in PCR amplifications that can also bind free magnesium (such as primers and template) therefore, the magnesium ion concentration should be titrated in order to find the optimum concentration for each reaction.

How much AmpliTaq DNA Polymerase is used in a PCR amplification?

Most PCR amplifications use 2.5 units of AmpliTaq DNA polymerase per 100 µL reaction. A 25 µL reaction would use about 0.6 units. However, the optimal unit concentration per reaction should be empirically determined, and often the less is better rule applies. Using too much AmpliTaq may result in non-specific amplification.

When amplifying long PCR targets, is the concentration of the deoxynucleoside triphosphates (dNTPs) limiting?

The concentration of dNTPs in a standard PCR amplification is 200 µM each, for a total of 800 µM. This total dNTP amount corresponds to 39 µg of dNTPs. This is a huge excess and, when generating long PCR fragments, is not a limiting factor during the PCR amplification, as the amount of target DNA generated is generally no more than 1 µg. More importantly, the reaction condition variables need to be monitored more closely in order for successful long PCR amplification to occur.

What thermal stable DNA polymerase is recommended for PCR amplification of long PCR targets?

Successful amplification of long PCR targets is dependent on variables such as sufficient extension time during the PCR amplification, cosolvent addition, pH of the reaction buffer, salt concentration, primer design, use of a hot start, DNA sample integrity, and the enzyme's proofreading and polymerase activities. A few examples of our long PCR enzymes include our Elonagase enzyme mix that can be used for amplicons up to 30kb (blend of Taq and proofreading enzyme) or our Phire Hot Start II enzyme mix that can be used for amplicons up to 20 kb (Taq polymerase). Read more here: https://www.thermofisher.com/us/en/home/life-science/pcr/pcr-enzymes-master-mixes/long-fragment-pcr.html

Does AmpliTaq Gold DNA Polymerase remain in an active state once it is activated?

Yes, once activated, AmpliTaq Gold DNA polymerase remains active. Lowering the temperature will not inactivate AmpliTaq Gold DNA polymerase.

Does the activation of AmpliTaq Gold DNA Polymerase at 95 degrees C for 10 min interfere with the half-life of the enzyme?

The half-life of AmpliTaq Gold Polymerase at 95 degrees C is 40 minutes. This is with constant incubation at the described temperature. During PCR, the reaction is only incubated at the programmed temperature for approximately 20 seconds. Therefore, the cycling half-life of AmpliTaq Gold Polymerase at 95 degrees C is approximately 100 cycles.

Example: AmpliTaq Gold DNA Polymerase experiences about 20 seconds at 95 degrees C per PCR cycle. The t_1/2 is at least 35 minutes; (35-40 min). Therefore, 35 min/20 sec/cycle = 105 cycles. 105 PCR cycles reduces enzyme activity by 50%.

Do AmpliTaq DNA Polymerase and AmpliTaq Gold DNA Polymerase add on extra A to the PCR product?

Both AmpliTaq Gold DNA polymerase and AmpliTaq DNA Polymerase lack proofreading activity, so they will often leave a 3'-overhang. The base most often left is a 3'-A, however, the extra base appears to be sequence dependent and one cannot always rely on the fact that even just a single base has been left. In many cases, this artifact has been useful with TA Cloning kits. In order to drive the reaction to the extra A state, a final extension time at 72°C should be increased to 15-30 minutes.

Why is AmpliTaq Gold Polymerase the enzyme of choice for multiplex PCR?

Multiplex PCR involves the coamplification of multiple amplicons in a single PCR. Since multiple sets of primers are being added to a single reaction, the potential for primer dimer formation as well as a general loss of specificity and a decreased yield of specific product exists. The ability to control the activation of AmpliTaq Gold Polymerase via hot start, so that the multiple primers do not have the possibility to react with themselves, has proven successful at alleviating these complications and dramatically increasing specific product yield.

Will PCR amplifications previously successful with regular AmpliTaq Polymerase work as successfully with AmpliTaq Gold Polymerase?

They will work more successfully and reproducibly with AmpliTaq Gold Polymerase. Although AmpliTaq Gold Polymerase is the same exact enzyme as AmpliTaq Polymerase, the fact that the reaction is being driven towards high specificity and yield may require some modifications to previous conditions. For example, if the previous reaction was on the edge of optimization, magnesium chloride concentrations may need to be re-optimized or if previous reactions were being run in a pH suboptimal for AmpliTaq Gold Polymerase, reaction conditions and sample preparation protocols may need to be revisited. Activation time for AmpliTaq Gold Polymerase will also need to be determined empirically and is dependent on cycler type.

Does AmpliTaq DNA Polymerase have exonuclease activities?

AmpliTaq DNA Polymerase lacks a 3' - 5' exonuclease activity. However, the enzyme does have a fork-like, structure-dependent polymerization-enhanced 5 ' - 3' nuclease activity. During the extension step of a PCR amplification, the enzyme will hydrolyze any blocking strand starting from its 5' end, replacing the lost material by extending the new chain.

Does AmpliTaq DNA Polymerase have reverse transcriptase activity?

Yes, AmpliTaq DNA Polymerase has been reported to exhibit reverse transcriptase activity, but it is such a low and inefficient activity that it is neither useful nor harmful to the RNA PCR experiments.