Lipofectamine™ 2000 Transfection Reagent, 1.5 mL - FAQs

我使用Lipofectamine 2000向细胞系中转染GFP，结果观察到明亮的颗粒状橙色背景荧光。这是什么原因所导致的？

使用阳离子脂质体转染可能会产生明亮的颗粒状橙色背景荧光。橙色荧光与脂质体/DNA复合物有关而与GFP无关。这一背景荧光随着所用阳离子脂质体试剂的不同而变化，但不会对转染结果造成干扰。如果需要，尝试在PBS中而不是培养基中进行荧光成像。同时，要确保细胞是健康的，完整的，因为溶解的细胞或在压力下的细胞可以产生自荧光产物。

我不慎将我的脂质体试剂留在了室温条件下，还能继续使用吗？

可以，我们所有的脂质体转染试剂均能够在室温下稳定保存数月。

我能否使用Lipofectamine RNAiMAX共转染siRNA和质粒DNA？

Lipofectamine 3000或Lipofectamine 2000可用于siRNA和质粒DNA的共转染实验，而Lipofectamine RNAiMAX并不适用。

我能否将Lipofectamine 2000用于质粒和siRNA的共转实验？

您可使用Lipofectamine 2000共转染质粒和siRNA。请点击此处（http://www.thermofisher.com/site/us/en/home/References/protocols/cell-culture/transfection-protocol/plasmid-co-transfection-protocol-lipofectamine-sirna.html）以获取转染方案相关信息。

Lipofectamine 2000:DNA复合物能够在多长时间内保持稳定？

使用Opti-MEM培养基配制Lipofectamine 2000:DNA复合物时，复合物在室温下6小时内保持稳定。如使用其他类型的培养基配制复合物，其稳定性可能稍低。

Lipofectamine 2000与Lipofectamine 2000 CD之间有何区别？

对Lipofectamine 2000 CD的CD设计最初是为了确保该试剂没有动物源性。然而，除另有说明外，我们所有的以脂质为基础的转染试剂都是无动物来源的。关于我们任何试剂的信息或问题都可联系我们的技术支持获得答案。

我应如何在Lipofectamine 3000，Lipofectamine 2000和Lipofectamine LTX之间做出选择？

Lipofectamine 3000是我们最好的转染试剂，强烈建议用其向各类细胞导入质粒DNA，其所适用的细胞系包括： - >60种细胞系（包括癌细胞系、成纤维细胞、神经细胞等） - 易转细胞 - 难转细胞（包括神经细胞、干细胞、血细胞） - 悬浮细胞 - 原代细胞 - iPSC 请参见我们的验证过的细胞系列表(https://www.thermofisher.com/us/en/home/life-science/protein-expression-and-analysis/transfection-selection/lipofectamine-3000.html?icid=fr-lipofectamine-2#validated)。 在某些种类的细胞中，Lipofectamine 2000与Lipofectamine LTX是可能达到更高效率的替代选项。需要解决细胞毒性问题时，Lipofectamine 3000是我们最为温和的试剂，其细胞毒性显著降低的同时，转染效能也有提升。Lipofectamine 3000和Lipofectamine 2000均可用于共转（siRNA与质粒DNA）实验。

反向转染与正向转染之间区别是什么？我该如何选择？

在正向转染过程中，细胞铺于培养孔之中，用通常的方式制备转染复合物并在第二天将其加入细胞培养物中。在反向转染过程中，在培养孔之中制备转染复合物，之后再加入细胞与培养基。反向转染比正向转染过程更迅速，因此是高通量转染的理想之选。在非高通量的转染操作中，通常正向转染对于大多数类型的细胞转染效果更佳。

你们建议使用何种转染试剂进行RNAi方面的应用？

我们建议您在所有细胞类型中使用Lipofectamine RNAiMAX试剂转染siRNA。该产品是专门针对siRNA转染而研发，能够提供高的转染效率和低的细胞毒性。因此，您只需进行些许的优化即可达到理想效果。针对基于载体DNA的RNAi应用，我们建议使用Lipofectamine 3000结合P3000增强剂进行转染。

转染过程中的培养基可使用抗生素么？

可以，转染过程中可在培养基中添加抗生素（青霉素-链霉素）。我们对多个细胞系在含与不含抗生素的培养基中的转染效果进行了比较，同时评估其转染效率和细胞毒性，结果显示并无差别。不过，某些细胞类型对转染过程敏感或可能出现细胞毒性方面的问题，此时省略抗生素可能会有助于改善结果。对稳定转染而言，转染操作后至少等待48小时以上，再加入选择性抗生素。

在脂质体转染过程中必须使用无血清的培养基么？

脂质体转染过程中不是必须采用无血清培养基。不过，脂质：核酸复合物一定要在无血清的条件下配制，因为（血清中的）蛋白质可能会干扰复合物的形成。一旦复合物形成，即可直接加至含血清培养基中的细胞中。

我应使用何种反应管来配制脂质体：DNA复合物？

聚丙烯、聚苯乙烯或玻璃管可与我们的任何转染产品一起使用而不会有任何问题。

你们建议使用何种脂质转染试剂来尝试转染我的细胞系？

我们建议使用Lipofectamine 3000进行质粒DNA转染，使用Lipofectamine MessengerMAX进行mRNA或短寡核苷酸转染，使用Lipofectamine RNAiMAX进行siRNA/miRNA的转染。请参考转染试剂选择指南(https://www.thermofisher.com/cn/zh/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html)并根据所用的细胞系和应用来进行正确的选择。

进行转染操作时，你们建议使用何种对照？

为了确保最佳转染成功，我们建议包括阳性转染对照和额外的对照来确认细胞健康和试剂质量。

对于DNA转染，我们建议使用pJTI R4 Exp CMV EmGFP pA载体(货号A14146)。对于siRNA转染，我们建议使用BLOCK-iT AlexaFluor红色荧光对照(货号14750100)或Silencer Select GAPDH阳性对照siRNA (货号4390850)。对于蛋白质转染，我们建议与EmGFP mRNA如Tri-Link CleanCap EGFP mRNA (货号L – 7201)共转染。

细胞健康和试剂质量对照包括：

纯细胞组
细胞+DNA或RNAi组
细胞+脂质试剂组
细胞+GFP质粒阳性对照组

为获得最佳转染效果使用脂质体转染试剂应注意的要点有哪些？

以下要点需要考虑：

1. 针对您的细胞类型来选择阳离子脂质试剂以获得最高转染效率。请参考转染试剂选择指南(http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html)进行正确的选择。
2. 优化阳离子脂质试剂和DNA的用量。除细胞状态以外最重要的因素是脂质体：DNA的比率。
3. 在复合物形成过程中不要使用血清。血清中可能含有能一直复合物形成的化合物。我们推荐使用Opti-MEM I减血清培养基以获得最佳的复合物形成效果。然而不含血清的DMEM或PRMI 1640也是可以使用的，但复合物形成效率可能不及Opti-MEM I减血清培养基那样高。
4. 在配制用于DNA-阳离子脂质体复合物形成的培养基中不要使用抗生素、EDTA、柠檬酸盐、磷酸盐、硫酸软骨素、透明质酸、硫酸葡聚糖或其他硫酸蛋白聚糖。
5. 转染时的细胞（密度）应处于60%至80%的汇合度。细胞应处于对数生长中期。为了确保在实验组间获得最为稳定的结果，建议您最好能够在实验开始时优化细胞铺板密度，而不是仅仅通过细胞汇合度来粗略估计。
6. 请确保转染DNA中的启动子-增强子能够兼容靶标细胞类型。
7. 不要使用冰冻过的，或在温度低于4°C的冰箱隔间中储存的阳离子脂质试剂。
8. 请在转染分析实验中加入阳性对照（比如选择货号 A14146 为追DNA转染对照选择货号 14750100 作为siRNA转染对照）。

同时，请参考此处(https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html)列举的要点提示。

你们的转染试剂的稳定性如何？

我们的转染试剂是在常温条件下运输的，在收到产品后应立即置于4℃条件下储存。如果贮存和使用得当，除了管子标签或者产品COA文件中有特别标注外，我们保证从收到产品之日起一年内产品的性能正常。我们不推荐冻存转染试剂，因为冻存一般会降低转染效果。

关于室温运输转染试剂的说明请参见此白皮书(http://tools.thermofisher.com/content/sfs/brochures/cms_103226.pdf)

我尝试了几种不同的转染试剂，但还是没能将我的基因转染进我所感兴趣的细胞系内。你们有何建议？

我们推荐您尝试使用电转法来转染您感兴趣的质粒进入细胞。我们提供Neon转染系统来帮助用户高效地转染原代细胞、干细胞以及一些难以转染的细胞。

您也可尝试使用病毒系统(https://www.thermofisher.com/us/en/home/life-science/protein-expression-and-analysis/protein-expression/mammalian-expression/viral-expression.html?icid=fr-viral-main%20http://www.lifetechnologies.com/us/en/home/life-science/protein-expression-and-analysis/protein-expression/mammalian-expression/viral-expression.html?icid=fr-viral-main)来转导您研究的基因进入到您感兴趣的哺乳动物细胞系中。

（通过显微镜）观察到在向细胞加入转染试剂：DNA复合物之后，细胞上出现了小颗粒状沉淀。这些沉淀是否会影响转染效率？

脂质体转染后在细胞上观察到沉淀的通常原因是体系中EDTA或阳离子脂质体过量。我们推荐用水稀释DNA，如果希望使用TE溶液，则EDTA的浓度应低于0.3 mM。同时请确保在复合物配制过程中，阳离子脂质体试剂的浓度不要超过推荐用量。细胞上这一沉淀物的存在与否与转染效率之间并无相关性。

为何转染之后我观察到细胞出现毒性反应？你们能提供一些帮助吗？

此处(https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html#2)是一些转染后细胞活性降低的可能原因及其相应的解决方案。请注意，最近研究发现，转染过程中可在培养基中使用抗生素。我们对多个细胞系在含与不含抗生素的培养基中的转染效果进行了比较，同时评估其转染效率和细胞毒性，结果显示并无差别。对稳定转染而言，转染操作后至少等待72小时以上，再加入选择性抗生素。

我实验的转染效率很低。你们能给予一些问题排查提示吗？

此处(https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html#1)收录了一些转染效率低的可能原因及其相应的解决方案。

如何制作剂量反应曲线或致死曲线？

剂量反应曲线是一种测定细胞接触不同浓度抗生素时的细胞毒性的有力工具。筛选抗性细胞所需的选择性抗生素的量因多种因素而异，包括细胞类型和抗生素类型。我们建议每次制作剂量-反应曲线时使用新的抗生素（或不同的品牌）或是使用不同的细胞系。
剂量-反应曲线测定的实验概要：

1. 将细胞铺在一定数目的孔中，使得它们有25-30％的汇合度。这表明细胞仍在分裂，并对抗生素反应良好。
2. 使用生长培养基稀释待测抗生素至推荐范围的广泛线性浓度。
3. 从细胞中移去生长培养基。将含抗生素的培养基加到各自孔中，留一组孔空置不加。在这些空孔中加入不含抗生素的生长培养基。
4. 在适当的生长条件下培养细胞（每隔3-4天更换一次培养基以去除死细胞，同时添加新鲜的含抗生素培养基）并每日观察细胞状态。在10-14天时，估算每孔中的活细胞数目。（这个时间周期取决于待测抗生素。抗生素如Geneticin、Hygromycin和Zeocin需要大约3周杀死细胞，因此等待10-14天较为理想。而Blasticidin杀死细胞需要大约2周，等待7-10天即可。）为了达到这一点，移去培养基，用磷酸盐缓冲盐水洗涤细胞并用0.5％的亚甲基蓝和50％甲醇进行细胞染色20分钟。
5. 将对应于不同抗生素浓度的活细胞数目绘图。这个曲线即是剂量-响应曲线或致死曲线。抗生素在所选时间段内杀死所有细胞的最低浓度就是接下来用于稳定筛选的浓度。

病毒转导相对于转染的主要优势在哪里？

转染无法用于某些细胞类型，例如非分裂细胞，而病毒转导既能用于分裂细胞又能用于非分裂细胞，比如难以转染的神经细胞。

相对于磷酸钙介导的转染，脂质体介导的转染的主要优势在哪里？

脂质体介导的转染的主要优势是能取得更高的转染效率，即使是不能使用磷酸钙介导转染的细胞类型。此外，脂质体介导的转染不仅能用于寡聚核苷酸到大DNA范围内的DNA递送，还能递送RNA和蛋白质。

瞬时转染和稳定转染的主要区别是什么？

对于瞬时转染，转染的DNA没有整合到宿主基因组中，所以外源DNA在后续的细胞有丝分裂阶段将丢失。外源基因的表达是短暂的（最长7-10天），但表达水平很高，因为在同一个细胞里会有多个拷贝的DNA。对于稳定转染，转染的DNA能够整合到宿主基因组，因此转染细胞及其子代细胞的基因组中会同时保留转染的DNA。外源基因也会随着筛选过程而表达。由于每个细胞中只整合了1-2个DNA拷贝，所以表达水平较低。稳定转染的转染效率只有瞬时转染的1-10％。

Lipofectamine3000与Lipofectamine2000一样吗？LipofectamineLTX、LipofectaminePLUS和Lipofectamine如何？

这些产品都是不同的阳离子脂质体试剂。Lipofectamine3000为质粒DNA和RNAi转入最广泛类型的细胞提供了最佳的转染性能。LipofectamineLTX用于以最小的细胞毒性递送质粒DNA。虽然PLUS试剂可以单独购买（货号11514-015），但LipofectaminePLUS转染试剂目前已经停产。Lipofectamine最初于20世纪80年代末发布，被认为是我们的第一代转染试剂。我们将继续为偏向使用老试剂的客户提供这些产品，但建议所有的新客户开始尝试Lipofectamine3000以获得最佳的转染性能和最低的细胞毒性。

如何根据A260读数来评估DNA的量？什么是A260/ A280？

1 A260单位（质粒DNA溶于H2O）=50mg/mL。如果质粒DNA使用除H2O之外其他缓冲液稀释，则消光系数会改变。这会导致上述值发生变化。
计算举例：
质粒DNA样品的体积=100mL
稀释（1/20）= 25mL样品加入475mLH2O中
稀释样品的A260=0.65
注：为了达到最佳效果，确保OD值在0.1和1.0之间。
质粒DNA样品的浓度=0.65×50mg/mL×20（稀释因子）=650mg/mL
样品的质粒DNA量=650mg/mL×0.1mL（样本量）= 65mg
A260 / A280值大于或等于1.8，意味着该质粒DNA是纯的；A260 / A280读数小于1.8，表明样品可能被芳香族产物（即苯酚）或蛋白质污染；读数大于2.0，则表明样品被RNA污染。

关于转染用无内毒素DNA的制备，有什么建议？

我们提供多种PureLinkHiPure纯化试剂盒来制备无内毒素DNA，PureLinkHiPure纯化试剂盒有Mini、Midi、Maxi、Mega和Giga几种规格。这些试剂盒包含一种采用阴离子交换树脂纯化质粒DNA的专利技术，该技术的纯化水平是氯化铯梯度纯化技术的两倍。想了解更多信息，请点击 此处(https://www.thermofisher.com/cn/zh/home/brands/product-brand/purelink.html)。 

我可以将质粒和siRNA共转染吗？

您可以使用i） Lipofectamine 3000和P3000一起 或是 ii）Lipofectamine 2000共转染质粒和siRNA。

什么类型的分子可以使用转染试剂转染？

我们的阳离子脂质体转染试剂可用于转染DNA、siRNA、Stealth RNAi、mRNA、dicer-产生的siRNA混合物、或shRNA质粒。寡核苷酸，蛋白质和RNA也可由其转染。DNA可以是质粒、粘粒，甚至是长达600 kb的YAC克隆。

我可以用你们的脂质体试剂来共转染质粒吗？

可以。标准转染实验方案需要保持转染混合物中DNA的总量恒定。也就是说，如果您的试验方案需要1 mg的质粒，则两种共转染质粒各使用0.5 mg，或是4种共转染质粒各使用0.25 mg。当利用共转染对不同的质粒引入选择性标记时，我们建议使用摩尔比3:1至10:1，目标质粒比选择质粒过量，以确保目标质粒和选择质粒同时存在。

哪里可找到使用过你们试剂的其他研究者的参考文献？

访问每种类型试剂的产品页面，在页面底部即可看到有一个引用列表。您也可访问一个罗列特定细胞系参考文献的表格。我们还建议采用www.highwire.org作为搜索引擎来查找使用我们转染产品的最新研究论文。您只需在搜索条件中直接列出转染试剂的名称和您的细胞系/应用。

在哪里可以找到特定细胞系的转染方案？

特定细胞系的转染方案可点击此处(https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support.html)找到。如果您未找到特定细胞系转染方案或者方案没有达到预期效果，我们建议测试随产品提供的实验方案中描述的条件，确定最佳的实验方案。 成功的转染依赖于细胞类型、脂质体用量、细胞的健康情况、传代次数和转染时的细胞密度。这些因素在各个实验室之间可能略有不同，为了得到相同的结果，可能需要对实验方案进行额外的优化。请访问我们的非常有用的转染实验问题排查技巧页面（https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html.），更多关于问题排查的信息欢迎访问我们的转染支持中心(thermofisher.com/transfectionsupport)。

为什么我的基因在瞬时转染的表达水平要高于稳定转染？

瞬时转染克隆的表达水平通常比较高，因为瞬时转染细胞通常具有较高的外源基因拷贝数（每个细胞几百个）。稳定转染的克隆通常有1-2个拷贝整合进基因组，因此其表达水平较低。有时候，稳定转染细胞的表达水平低是因为当组成型表达重组蛋白时对细胞造成了负面影响。

为什么我的转染实验不具有可重复性？

一般情况下，无论如何尝试控制转染影响因素，两次转染的效率仍会表现出一定程度的差异性。转染时，请保持所有转染影响因素，如细胞汇合度、传代次数和生长期一致。尽可能解冻新的细胞。为了尽量减少转染差异的影响，可以使用内参，如β半乳糖苷酶或萤光素酶。可以共转染表达质粒和参考质粒，分析β-gal或荧光素酶的活性。

我使用的孔的大小与您在试验方案中详细描述的有所不同。该如何放大或缩小我的转染体系呢？

我们的每个转染试剂实验方案均提供了一个表格，用于放大或缩小转染体系。详细信息请参考具体的说明书。

我可以针对不同的细胞系使用相同量的任意转染试剂吗？

不可以，转染效率和每个孔所用试剂的量密切相关，并可能因试剂而异。为了获得最佳转染效率，请参考随转染试剂提供的产品信息。
随产品提供的实验方案将为您提供每个孔转染试剂的最佳使用量范围。在产品开发期间，该使用量在多种细胞系中都能很好地工作。如果在您特定的细胞系中没有得到预期的效果，可能还需要做进一步的优化。请访问我们的非常有用的转染实验问题排查技巧页面（https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html.），更多关于问题排查的信息欢迎访问我们的转染支持中心(thermofisher.com/transfectionsupport)。

细胞密度（％汇合度）和传代次数是转染需要重要考虑的因素吗？

是的，细胞密度会影响转染性能。 Lipofectamine 3000, Lipofectamine 2000和 Lipofectamine LTX/PLUS能够在70-90％的细胞汇合度时展现优异的转染性能，而汇合度低于这一数值时，则可能观察到细胞毒性。Lipofectamine RNAiMAX在60-80％的细胞汇合度时效果最佳。
传代次数可能会影响转染实验。我们推荐一致地传代和接种细胞。传代次数过多可能会降低转染效率。我们不推荐超过20-30次传代的细胞。如果转染性能下降并且细胞已经培养过很长时间或过度/不当传代，我们建议您从液氮中复苏一瓶新细胞重新培养。请参阅 Gibco细胞培养基础手册(https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics.html)，了解正确培养和传代细胞的指南。

关于转染试剂的选择，你们有什么建议？

请参阅转染试剂选择指南(https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html)进行正确的选择。

转染有哪些不同的方法？

目前有很多种基因递送技术，可向真核细胞中导入质粒DNA、siRNA或双链RNAi、寡核苷酸和RNA，以便进行各种研究和药物开发应用。此处(https://www.thermofisher.com/cn/zh/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html)提供了有关这些技术及其优缺点的综述。

I transfected GFP into cells using Lipofectamine 2000 and saw a light granular orange background fluorescence. What could be causing this?

Transfection with cationic lipids can produce light granular orange background fluorescence. The orange fluorescence is associated with the lipid/DNA complexes and is not related to GFP. This background varies depending on the cationic lipid reagent used and does not interfere with transfection results. If desired, try performing fluorescence imaging in PBS instead of culture medium. Also, make sure the cells are healthy and intact as lysed cells or cells under stress could generate autofluorescence products.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

I accidentally left my lipid reagent at room temperature. Can I still use it?

Yes, all of our lipid transfection reagents are stable at room temperature for months.

Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

Can I use Lipofectamine RNAiMAX to co-transfect siRNA with plasmid DNA?

While Lipofectamine 3000 or Lipofectamine 2000 may be used for co-transfection of siRNA with plasmid DNA, Lipofectamine RNAiMAX cannot be used.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I use Lipofectamine 2000 to co-transfect plasmids and siRNA?

You can co-transfect plasmids and siRNA using Lipofectamine 2000. Please click here (http://www.thermofisher.com/us/en/home/references/protocols/cell-culture/transfection-protocol/plasmid-co-transfection-protocol-lipofectamine-sirna.html) for protocol information.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

For how long is the Lipofectamine 2000:DNA complex stable?

Lipofectamine 2000:DNA complexes are stable for 6 hours at room temperature when the complex is prepared in Opti-MEM medium. Use of other media for complex formation may decrease stability.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the difference between Lipofectamine 2000 and Lipofectamine 2000 CD?

The CD designation for Lipofectamine 2000 CD was originally provided to ensure this reagent is animal origin-free. However, all of our premium Lipofectamine lipid-based transfection reagents are animal origin-free unless stated otherwise. Please contact Technical Support for information or questions regarding any of our reagents.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How do I choose between Lipofectamine 3000, Lipofectamine 2000, and Lipofectamine LTX?

Lipofectamine 3000 (Cat. No. L3000015is our best transfection reagent and we highly recommend this over all other reagents for plasmid DNA delivery for a broad range of cell types which includes:

- 60 cell lines (i.e., cancer, fibroblasts, neural, etc.)
- Easy-to-transfect cells
- Hard-to-transfect cells (i.e., neural, stem, blood-derived)
- Suspension cells
- Primary cells
- iPSCs
Please see our table of validated cell lines (https://www.thermofisher.com/us/en/home/brands/product-brand/lipofectamine/lipofectamine-3000.html).

Lipofectamine 2000 (Cat. No. 11668019) and Lipofectamine LTX (Cat. No. 15338100) are alternative options that may work better for certain cell types. For addressing cytotoxicity issues, Lipofectamine 3000 is our most gentle reagent with significantly reduced cytotoxicity levels and improved transfection performance. Both Lipofectamine 3000 and Lipofectamine 2000 may be used for co-transfection (siRNA and plasmid DNA) experiments.

Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

What is the difference between reverse transfection and forward transfection? What should I use?

In forward transfection, cells are seeded to appropriate confluence or cell density in wells or dishes, and the lipid-DNA complexes are added the next day. In reverse transfection, the transfection complexes are prepared inside the wells, after which cells and medium are added. Reverse transfection is faster to perform than forward transfection, and is the method of choice for high-throughput transfection. For non-high-throughput transfections, generally forward transfections have better efficiency for most cell types.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Which transfection reagent do you recommend for RNAi applications?

We recommend using Lipofectamine RNAiMAX Reagent (Cat. No. 13778150) for delivery of siRNA and miRNA into all cell types. It has been specifically developed for siRNA transfection while providing high transfection efficiency with minimal cytotoxicity. As a result, less optimization is necessary. For vector DNA-based RNAi applications, we recommend Lipofectamine 3000 Reagent (Cat. No. L3000015) with the P3000 Enhancer Reagent. For CRISPR-mediated gene knockout methods, we recommend Lipofectamine CRISPRMAX (Cat. No. CMAX00015) for Cas9/gRNA ribonucleoprotein, Lipofectamine MessengerMAX (Cat. No. LMRNA001) for Cas9 mRNA, and Lipofectamine 3000 (Cat. No. L3000015) with the P3000 Enhancer Reagent for CRISPR plasmid DNA-based delivery. For all difficult-to-transfect cells, we recommend using electroporation methods such as the Neon Transfection System (Cat. No. MPK5000).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Is there a place where I can find references from other researchers who have used your transfection reagents?

Visit the product page for each reagent type and you will see a list of references at the bottom of the page. A table that lists specific cell line references is also accessible. We also recommend www.highwire.org as a search engine to find a large selection of up-to-date research articles using our transfection products. Simply include the name of the transfection reagent and your cell line/application of interest in your search criteria.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I use antibiotics in the medium during transfection?

Antibiotics can be used in the medium for culturing of cell lines. However, we do not recommend using antibiotics in the transfection medium unless previously tested in the cell type and payload being transfected. This is because presence of antibiotics during transfection may adversely affect transfection efficiency (i.e., positively charged antibiotics binding to the DNA being transfected) and overall health of cells being transfected.

For stable transfection, we recommend waiting wait 24-48 hrs after transfection before adding selected antibiotics.

Find additional tips, troubleshooting help, and resources within ourTransfection Basics Support Center.

Is it necessary to use serum-free medium during lipid transfection?

It is not necessary to use serum-free medium during lipid transfection. However, it is critical to form the lipid:nucleic acid complex in the absence of serum, because proteins can interfere with complex formation. Once the complexes are formed, they can be added to cells in serum-containing medium. For optimal results with Lipofectin Transfection Reagent, we recommend performing transfection in medium without serum.

Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

What kind of tubes should I use for making lipid:DNA complexes?

Polypropylene, polystyrene, or glass tubes may be used with any of our transfection products without issue.

Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

Which lipid transfection reagent would you recommend to use for my cell line?

We recommend using Lipofectamine 3000 Reagent (Cat. No. L3000015) for the delivery of plasmid DNA, Lipofectamine MessengerMAX Reagent (Cat. No. LMRNA001) for mRNA or short oligos, and Lipofectamine RNAiMAX Reagent (Cat. No. 13778150) for siRNA or miRNA. Please refer to the Transfection reagent selection guide (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html) to choose the best reagent based on cell type and application.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What controls do you recommend that I use when performing a transfection?

To ensure optimal transfection success, we recommend including a positive transfection control and additional controls to confirm cell health and reagent quality.

For DNA transfection, we recommend using the pJTI R4 Exp CMV EmGFP pA Vector (Cat. No. A14146). For siRNA transfection, we recommend using either the BLOCK-iT AlexaFluor Red Fluorescent Control (Cat. No. 14750100) or Silencer Select GAPDH Positive Control siRNA (Cat. No. 4390850). For protein transfection, we recommend co-transfecting with an EmGFP mRNA such as the Tri-Link CleanCap EGFP mRNA (Cat. No. L-7201).

Cell health and reagent quality controls:
- Cells only
- Cells + DNA or RNA or protein only
- Cells + lipid reagent only
- Cells + Opti-MEM only
- Cells + positive control

Find additional tips, troubleshooting help, and resources within ourTransfection Support Center.

What points should I consider to achieve optimal transfection with a lipid-based transfection reagent?

Here are some points to consider:

1. Select the lipid reagent that is likely to result in highest transfection efficiency for your cell type, payload, and application. Please refer to the Transfection Reagent Selection Guide (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html) to choose the best reagent.
2. Optimize both lipid reagent and DNA quantities. The most important parameter after the condition of the cells is the ratio of lipid to DNA.
3. Do not use serum during complex formation. Serum may contain components that could interfere with complex formation. We recommend using Opti-MEM I Reduced-Serum Medium for optimal complex formation. However, serum-free DMEM or serum-free RPMI 1640 Medium can be used, but the efficiency of complex formation may not be as high as with Opti-MEM I Reduced-Serum Medium.
4. Do not use antibiotics, EDTA, citrate, phosphate, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the medium used to prepare the DNA-cationic lipid reagent complexes.
5. Cell density should be between 50% to 80% confluency at the time of transfection (please refer to specific reagent manual for details). Cells should be in the mid-log growth phase. For better consistency and reproducibility of results between transfection experiments, accurately count your cells with either a hemocytometer or the Countess II FL Automated Cell Counter (Cat. No. AMQAF1000).
6. Confirm that the promoter and/or enhancer (any gene regulatory sequences) of the transfected DNA is compatible with the target cell type.
7. Do not use a cationic lipid reagent that has been frozen or stored at temperatures below 4 degrees C.
8. Include a positive control for the transfection assay (for example, Cat. No. A14146 for plasmid DNA transfection and Cat. No. 14750100 for siRNA transfection).

For additional tips ,please take a look at the tips outlined here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html).

Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

What is the stability of your transfection reagents?

Our transfection reagents are shipped under ambient conditions and should be stored at 4 degrees C immediately upon receipt. We guarantee the performance of the product, if stored and handled properly, for one year from date of shipment unless otherwise stated on the tube label or COA. We do not recommend freezing transfection reagents, as this usually decreases transfection performance.

Please see this white paper (http://tools.thermofisher.com/content/sfs/brochures/cms_103226.pdf) on ambient shipping of Lipofectamine transfection reagents.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

I have tried several different transfection reagents and have failed to transfect my gene into my cell line of interest. Do you have any suggestions?

We recommend that you try electroporation as a method of delivering your plasmid of interest. We offer the Neon Transfection System for highly efficient transfection of primary cells, stem cells, and difficult-to-transfect cells. You may also consider using a viral-based system (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-expression/mammalian-protein-expression/viral-delivery-mammalian-expression.html) to deliver your gene into your mammalian cell line of interest.

Find additional tips, troubleshooting help, and resources within ourTransfection Basics Support Center.

I see a small granular precipitate on my cells (microscopically) following addition of the transfection reagent:DNA complexes to my cells. Will it decrease the transfection efficiency?

It is normal to see some precipitate on the cells. However, a common reason for detecting unsually high precipitate on cells following lipid-based transfection is if there is excess EDTA or cationic lipid present. We recommend diluting the DNA in water or, if TE is preferred, use EDTA concentrations of <0.3 mM. Also ensure that concentrations of cationic lipid reagents do not exceed recommended amounts during complex formation. The presence or absence of this precipitate is not indicative of the transfection performance.

Find additional tips, troubleshooting help, and resources within ourTransfection Basics Support Center.

Why do I see cytotoxicity after performing transfection with Lipofectamine 2000? Can you please help?

Below are possible reasons why you may see reduced viability following transfection, along with suggested solution.

1. Possible Cause: DNA: transfection reagent ratio sub-optimal for cell line
   Suggested Solution: Prepare complexes using a DNA (µg) to Lipofectamine 2000 (µl) ratio of 1:2to 1:3 for most cell lines. Optimization may be necessary. If so, vary DNA (µg): Lipofectamine 2000 (µl) ratios from 1:0.5 to 1:5.
2. Possible Cause: Plasmid DNA preparation contains high levels of endotoxin
   Suggested Solution: Ensure that the plasmid DNA or siRNA used for transfection is of high quality. For plasmid DNA purification kits, we recommend using our PureLink HiPureNucleic Acid Purification Kits.
3. Possible Cause: Cell density was not optimal
   Suggested Solution: Lipofectamine 2000 works best in cultures that are >90% at the time of transfection.
4. Possible Cause: Complexes were added to cells in serum-free medium
   Suggested Solution: Try using growth medium containing serum when performing transfections. Transfection performance is typically better when the cells are more viable. If you require serum-free conditions, test medium for compatibility with Lipofectamine 2000 since some serum free formulations (e.g. CD293, SFM II, VP-SFM) may inhibit cationic lipid-mediated transfection.
5. Possible Cause: Complexes not thoroughly mixed in growth medium
   Suggested Solution: Following addition of transfection complexes into medium, ensure that the plate or wells are thoroughly mixed to prevent concentration of DNA:transfection reagent complexes in the wells
6. Possible Cause: Cells have changed over time, or splitting conditions have changed
   Suggested Solution: If transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
7. Possible Cause: Antibacterial agents were used in growth medium during transfection
   Suggested Solution: Do not use antibiotics such as chloroquine, penicillin, or streptomycin in growth medium because during transfection, cells are more permeable to antibiotics, which may cause toxicity.
8. Possible Cause: Transfection reagent stored improperly
   Suggested Solution: We recommend storing transfection reagents at 4°C. Freezing of transfection reagents, or storing them at room temperature, may decrease activity.
9. Possible Cause: Cationic lipid reagent was oxidized
   Suggested Solution: Do not vortex or agitate cationic lipid reagents excessively; this may form cationic lipid reagent peroxides.
10. Possible Cause: Selection antibiotic added too soon
    Suggested Solution: When creating stable cell lines, allow at least 72 hr for cells to express the resistance gene before adding selective antibiotic.

I am getting very low transfection efficiency with Lipofectamine 2000. Can you please provide some troubleshooting tips?

Below are possible reasons why you may be getting low transfection efficiency, along with suggested solutions:

1. Possible Cause: Plasmid DNA, siRNA, or transfection reagent diluted in media containing serum or complexes formed in the presence of serum
   Suggested Solution: Use serum-free medium for dilutions of plasmid DNA, siRNA, and transfection reagents. Note: we recommend using Opti-MEM | Reduced Serum Medium (Cat. No. 31985-062)to dilute Lipofectamine 2000 and DNA before complexing.
2. Possible Cause: DNA: transfection reagent ratio sub-optimal for cell line
   Suggested Solution: Prepare complexes using a DNA (µg) to Lipofectamine 2000 (µL) ratio of 1:2 to 1:3 for most cell lines. Optimization may be necessary. If so, vary DNA (µg): Lipofectamine2000 (µL) ratios from 1:0.5 to 1:5. If using a different transfection reagent, please consult the product manual.
3. Possible Cause: Not enough plasmid DNA used for dilution or complex formation
   Suggested Solution: Verify concentration using a second method or check the DNA for degradation. Determine DNA concentration by performing A260/A280 readings on a spectrophotometer or by using the Quant-iT DNA Assays Kits (Q33130, Q33120).
4. Possible Cause: Plasmid DNA or siRNA used in transfection has degraded or is of poor quality
   Suggested Solution: Ensure that the plasmid DNA or siRNA used for transfection is of high quality. For plasmid DNA purification kits, we recommend using our PureLink HiPure Nucleic Acid Purification Kits.
5. Possible Cause: Cell density was not optimal
   Suggested Solution: Lipofectamine 2000 works best in cultures that are >90% at the time of transfection.
6. Possible Cause: Complexes were added to cells in serum-free medium
   Suggested Solution: Try using growth medium containing serum when performing transfections. Transfection performance is typically better when the cells are more viable. If you require serum-free conditions, test medium for compatibility with Lipofectamine 2000 since some serum-free formulations (e.g. CD293, SFM II, VP-SFM) may inhibit cationic lipid-mediated transfection.
7. Possible Cause: Inhibitors were present in medium
   Suggested Solution: Do not use antibiotics, EDTA, citrate, phosphate, RPMI, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the growth medium or in the medium used to prepare DNA:transfection reagent complexes.
8. Possible Cause: Problems with assay used to measure efficiency or expression
   Suggested Solution: Use a reporter gene to measure transfection efficiency. A reporter gene control allows you to confirm expression.
9. Possible Cause: Promoter-enhancer on vector is not recognized by the cell type
   Suggested Solution: Verify that the promoter-enhancer on your vector construct is compatible with the target cell type.
10. Possible Cause: Cells have changed over time, or splitting conditions have changed
    Suggested Solution: If transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
11. Possible Cause: Transfection reagent stored improperly
    Suggested Solution: We recommend storing transfection reagents at 4°C. Freezing of transfection reagents, or storing them at room temperature, may decrease activity.


Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

How do I perform a dose-response curve or kill curve?

The dose-response curve is a valuable tool to determine cell toxicity when exposed to various concentrations of antibiotic. The amount of selective antibiotic required to select for resistant cells varies with a number of factors, including cell type and type of antibiotic. We recommend performing a dose-response curve every time a new antibiotic (or a different brand) or a different cell line is used.

Experimental outline of dose-response curve assay:

1.Plate cells in a number of wells such that they are 25–30% confluent. This means that the cells are still dividing and hence will respond well to the antibiotic.
2.Dilute the antibiotic being tested to a broad linear concentration of the recommended range in growth medium.
3.Remove the growth medium from the cells. Apply the antibiotic-containing medium to the respective wells, leaving one set of wells empty. To these wells, add growth medium that does not contain the antibiotic.
4.Culture cells under proper growth conditions (change the medium every 3–4 days to get rid of dead cells and add fresh medium containing antibiotic) and observe the cells daily. At 10–14 days, assess the number of viable cells in each well. (This time period depends upon the antibiotic being tested; antibiotics such as Geneticin, Hygromycin, and Zeocin take about 3 weeks to kill cells, so waiting for 10–14 days would be ideal. However, for Blasticidin, which kills cells in about 2 weeks, waiting for 7–10 days would be sufficient.) To do this, aspirate the medium, wash the cells with phosphate-buffered saline and stain the cells with 0.5% methylene blue and 50% methanol for 20 minutes.
5.Plot the number of viable cells against the antibiotic concentration. This curve is the dose-response curve or kill curve. The lowest concentration of the antibiotic that kills all the cells in the chosen time period is then used for the stable selection.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the main advantage of viral transduction over transfection?

Transfection does not work for certain cell types such as non-dividing cells, whereas viral transduction works for dividing as well as non-dividing cells, such as neuronal cells that are hard to transfect.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the main advantage of lipid-mediated transfection over calcium phosphate-mediated transfection?

The main advantage of lipid-mediated transfection is the higher transfection efficiency that can be achieved with cell types that cannot be transfected using calcium phosphate. Calcium phosphate is prone to variability due to its sensitivity to slight changes in pH, temperature, and buffer salt concentrations. Calcium phosphate may also be cytotoxic to many cell types, especially primary cells. Further, lipid-mediated transfection can be used to deliver DNA ranging from oligos to large DNA, and can also deliver RNA and protein.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the main difference between transient and stable transfection?

During transient transfection the exogenous DNA does not integrate into the host genome, as a result some DNA is lost with every subsequent cell division. The expression is short-lived (maximum of 7-10 days) but the level of expression is high, since up to hundreds of copies of the DNA may be delivered into the cell. In stable transfection, under antibiotic selection pressure, the DNA integrates into the host cell genome and is passed onto their daughter cells during cell division. The expression is thus sustained as long as the selection pressure is maintained. The expression level is low since only 1-2 copies of the DNA may be integrated per cell. Transfection efficiency in a stable transfection is about 1-10% of that in a transient transfection.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Is Lipofectamine 3000 the same as Lipofectamine 2000? What about Lipofectamine LTX, Lipofectamine PLUS, and Lipofectin?

All of our lipid reagents have different cationic-lipid formulations, each with unique properties and specific applications. Lipofectamine 3000 (Cat. No. L3000015) provides the best transfection performance with lowest cytotoxicity for plasmid DNA and RNAi delivery for all cell types. Lipofectamine LTX (Cat. No. 15338100) was formerly designed for delivery of plasmid DNA with minimal cytotoxicity. Lipofectamine PLUS is a discontinued transfection reagent, although the PLUS Reagent is available and sold separately (Cat. No. 11514-015). Lipofectin (Cat. No. 18292011) was originally launched in the late 1980s and is considered our very first transfection reagent. We continue to offer these products for customers who prefer the older formulations, but recommend that all new users try Lipofectamine 3000 first for optimal performance and lowest toxicity.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How do you estimate the amount of DNA using A260 readings? What does A260/A280 mean?

1 A260 unit (double stranded DNA in H2O) = 50 mg/mL. The extinction coefficient will change if DNA is diluted in a buffer other than H2O. This will change the value indicated above.

Sample calculation:

Volume of plasmid DNA sample = 100 mL

Dilution (1/20) = 25 mL of the sample in 475 mL H2O

A260 of diluted sample = 0.65

Note: For optimal results, make sure OD values are within 0.1 and 1.0.

Concentration of plasmid DNA sample = 0.65 x 50 mg/mL x 20 (dilution factor) = 650 mg/mL

Amount of plasmid DNA in sample = 650 mg/mL x 0.1 mL (sample volume) = 65 mg

An A260/A280 value that is between 1.8 and 2.0 means that the plasmid DNA is pure. A260/A280 readings that are less than 1.8 indicate that the sample may be contaminated with aromatic products (i.e., phenol) or protein. Readouts greater than 2.0 suggest that the sample is contaminated with RNA.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What are your recommendations with regards to endotoxin-free DNA for transfections?

For transfection-grade plasmid DNA, use either a PureLink HiPure or PureLink Expi Plasmid Purification Kit with plasmid purity equivalent to 2X CsCl gradients. For endotoxin-free DNA (< 9.1 EU/µg) use a PureLink Expi Endotoxin-Free Kit (Maxi, Mega, or Giga). Endotoxin-free DNA is recommended for sensitive applications such as transfection of primary cells and research on gene therapy for plasmid vaccines. For more information, please click here: http://www.thermofisher.com/us/en/home/brands/product-brand/purelink.html.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I co-transfect plasmids and siRNA?

Yes. You may co-transfect plasmids and siRNA using i) Lipofectamine 3000 together with P3000 or ii) Lipofectamine 2000.

Find additional tips, troubleshooting help, and resources within ourTransfection Basics Support Center.

What types of molecules can be transfected using your lipid-based transfection reagents?

Our cationic lipid transfection reagents are used to transfect DNA (plasmids or oligonucleotides), siRNA (or miRNA), mRNA, or proteins. DNA delivered may be in the form of plasmids, cosmids, or even YAC clones as large as 600 Kb. Please refer to the Transfection reagent selection guide (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html) to choose the best reagent based on cell type and application.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I use your lipid reagents to co-transfect plasmids?

Yes. The standard transfection protocol may be followed by keeping the total amount of DNA in the mixture constant. That is, if your protocol requires 1 µg plasmid, use 0.5 µg of each of two co-transfected plasmids, or 0.25 µg of each of 4 co-transfected plasmids. When performing co-transfections to introduce a selectable marker on a different plasmid, we recommend using a 3:1 to 10:1 molar excess of the plasmid of interest over the selectable plasmid to ensure that the plasmid of interest is present with the selectable plasmid.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Is there a place where I can find references from other researchers who have used your reagents?

Visit the product page for each reagent type and you will see a list of references at the bottom of the page. A table that lists specific cell line references is also accessible. We also recommend www.highwire.org as a search engine to find a large selection of up-to-date research articles using our transfection products. Simply include the name of the transfection reagent and your cell line/application of interest in your search criteria.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Where can I find cell line-specific transfection protocols?

Cell line-specific transfection protocols can be found here (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/transfection-selection-tool.html). If you do not find a cell line-specific protocol or if the transfection does not perform as expected, we recommend optimizing the conditions described in the product manual. Successful transfection depends on the cell type, amount of lipid, cell health, passage number, and cell density at the time of transfection. Each of these factors may differ slightly from lab to lab and may require additional optimization of the protocol to achieve the same result. Please review our helpful troubleshooting tips: https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html. For more troubleshooting tips, please visit our Transfection Support Center (thermofisher.com/transfectionsupport).

Find additional tips, troubleshooting help, and resources within ourTransfection Basics Support Center.

Why would the expression level of my gene in transiently transfected cells be greater than those that are stably transfected?

Expression in transiently transfected clones is typically higher because transiently transfected cells can have up to hundreds of copies of the plasmid containing the gene of interest. Stably transfected clones usually harbor 1-2 copies integrated into the genome, and hence have lower levels of expression. Sometimes, the lower expression level in stably transfected cells is due to adverse effects of the recombinant protein on the cell when expressed constitutively.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Why are my transfections not reproducible?

In general, transfection efficiency will show some degree of variability between transfection experiments and among replicates in the same transfection experiment. For better reproducibility, keep all transfection parameters, such as cell confluency, passage number, and phase of growth, consistent between transfections. If possible, thaw fresh cells. We recommend preparing one master mix of the DNA/lipid complexes for the number of transfections planned to reduce multiple pipetting errors. When adding your complexes, we recommend changing tips between wells since re-used tips could bring carryover, especially for the 96- or 384-well format with small-volume formats. To further minimize the effects of transfection variability on data analysis, consider co-transfecting an internal normalization reference control such as beta-galactosidase or luciferase with the expression plasmid. Below are possible reasons for why your transfection results are not reproducible, along with suggested solutions:

1. Possible Cause: Cells have changed over time, or splitting conditions have changed
   Suggested Solution: If transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
2. Possible Cause: Transfections performed at different cell confluencies, or at different DNA:transfection reagent ratios
   Suggested Solution: Transfection performance reproducibility is dependent on day-to-day consistency in cell splitting, plating and transfecting with a consistent protocol (same DNA:transfection reagent ratios). Different DNA preparations or media changes may also change transfection performance.

I am working with well sizes different from those specified in your protocol. How do I scale up or scale down my transfection reaction?

Each of our transfection reagent protocols provides a table for scaling up or down transfections. Please consult the specific manual for details. For well or plates sizes not listed in the scaling table, calculate the total surface area and estimate the -fold difference from the 24-well. Use this -fold difference to adjust for reagent volumes, payload quantities, and seeding densities.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I use the same amount of any transfection reagent for different cell lines?

No.The transfection efficiency is highly dependent on the amount of reagent used per well and may be different between reagents. Please consult the product information that is provided with the transfection reagent for optimal use.

The protocol that is supplied with the product will provide you with an optimal range of transfection reagent to use per well. During product development, this range was determined to work well across a variety of cell lines. If you are still not achieving the performance you desire in your particular cell line, further optimization may be necessary. Please review our helpful troubleshooting tips: https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html. For additional troubleshooting tips, please visit our Transfection Support Center (thermofisher.com/transfectionsupport)

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Are cell density (% confluency) and passage number important considerations for transfection?

Yes, cell density is an important parameter in influencing transfection efficiency. If the seeding density is too low, some cytoxicity may be observed. If the cell density is high, lower than expected transfection efficiency may be observed. Both issues may be easily resolved by either descreasing or increasing the quantity of complexes added to the culture. We recommend using Lipofectamine 3000 since it shows the best flexibility for variable seeding density without showing cytotoxicity issues and maintains high protein expression. Lipofectamine 3000, Lipofectamine 2000, and Lipofectamine LTX/PLUS provide excellent transfection efficiencies at confluencies between 70 and 90%. Some toxicity may be observed at lower confluencies but may be alleviated by decreasing quantity of complexes or removing the complexes after 4-6 hours incubation and refreshing the media. Lipofectamine RNAiMAX works best at confluencies between 60 and 80%.

Passage number may affect transfection experiments. We recommend consistent splitting and plating of cells. Excessive numbers of passages may decrease transfection performance. We do not recommend splitting cells for more than 20-30 passages. If transfection performance declines and cells have been in culture for a long time or excessively/improperly passaged, we recommend that you restart your cultures with a new vial of cells from liquid nitrogen. Please refer to the Gibco Cell Culture Basics handbook (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics.html) for proper guidelines for culturing and passaging cells.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What recommendations do you have for selecting a transfection reagent?

Choose the best reagent by cell type and application by using the Transfection reagent selection guide (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What are the different methods available for transfection?

There are many transfection methods available to deliver plasmids, DNA fragments, oligos, siRNAs, mRNA, or proteins for a wide range of research and drug discovery applications. A review of the pros and cons of each technique is provided here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/gene-delivery-selection-guide.html).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How does FreeStyle MAX reagent compare to Lipofectamine 2000 and 293fectin reagents?

Lipofectamine 2000 is designed for adherent cells. FreeStyle and 293fectin reagents are designed for suspended Hek293 and CHO cells. For transfecting 293 suspension cells, FreeStyle MAX reagent yields transfection efficiency comparable to Lipofectamine 2000 and 293fectin reagents. However, Lipofectamine 2000 and 293fectin reagents do not work well for CHO suspension cells.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How do I choose between Lipofectamine 2000 and Lipofectamine LTX?

Lipofectamine LTX (Cat. No. 15338100) and Lipofectamine 2000 (Cat. No. 11668019) are both recommended for plasmid DNA transfections. However, if you notice increased cytotoxicity with Lipofectamine 2000, Lipofectamine LTX is the preferred reagent due to significantly reduced cytotoxicity levels and improved transfection performance in a number of hard-to-transfect cell lines.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Which lipid transfection reagent do you recommend for my cell line?

The best transfecting agent and efficiency would depend on the particular cell line you have. Please visit our online selection tools to see our recommendation for your specific cell line. If your cell line is not on the list, we recommend you try Lipofectamine LTX (Cat. No. 15338100) or Lipofectamine 2000 (Cat. No. 11668019) for plasmid transfection, and Lipofectamine RNAiMAX (Cat. No. 13778150) for siRNA transfection. For primary cells, Lipofectamine LTX with PLUS reagent (Cat. No. 15338100 and Cat. No. 11514-015) is generally the best choice for plasmid transfection. For some hard-to-transfect cells, like suspension cells and stem cells, the Neon electroporation system (Cat. No. MPK5000) usually works better compared to lipid transfection reagent.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Are lipid transfection reagents fluorescent?

The intrinsic fluorescence of Lipofectamine 2000 and Lipofectamine under FITC conditions was examined and the results were as follows:
- Lipofectamine 2000 in serum-free conditions (with or without DNA) - weak whitish fluorescence.
- Lipofectamine 2000 in serum-containing conditions (with or without DNA) - medium white and red fluorescence.
- Lipofectamine & Lipofectamine PLUS (with or without DNA) - orange fluorescence.
- We haven't seen any significant green fluorescence attributable to the Lipofectamine.

These observations were made after transfection of CHO-K1 cells and by examining the live cells in PBS. Other lipid transfection reagents were not studied extensively on this issue, but it is very likely they will produce fluorescence in transfected cells.

Fluorescence from various media components may be where the problem lies. For example, riboflavin apparently fluoresces in the green wavelength and thus DMEM, which has a high riboflavin content, can be problematic in fluorescence experiments. Ham's F12 medium is 10x lower in riboflavin and is more suitable for fluorescence work.

One possible solution is to perform fluorescence imaging in PBS, rather than in culture medium. Also, check to make sure the cells are healthy and intact. Lysed cells or cells under stress may generate autofluorescent products.


Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How can I optimize transfection conditions for Lipofectamine 2000?

Optimization of transfection conditions is essential for the highest-efficiency transfections and the lowest toxicity. The conditions that should be optimized include Lipofectamine 2000 and DNA concentrations, and cell number. It is recommended that conditions be tested and optimized for cell lines which do not have cell specific protocols available.

To optimize the amount of Lipofectamine 2000 reagent, start with cells at >90% confluency and 0.8-1.2 µg DNA for 24-well plates. With cell number and DNA concentration held constant, vary the amount of Lipofectamine 2000 Reagent to determine the optimal concentration (usually 1.5-3 µl). The cell number and amount of DNA can also be optimized. It is possible to minimize the effect of transfection on cell growth and viability by increasing the number of cells plated per well or by decreasing either Lipofectamine 2000 reagent or DNA concentrations. With careful optimization this can be achieved with little impact on the level of transgene expression.


Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How should I store Lipofectamine 2000?

- Lipofectamine 2000 should be stored at 4°C. When stored at 4°C, the reagent is guaranteed stable for one year from date of shipment.
- Lipofectamine 2000 should never be frozen. The reagent may survive one accidental freeze-thaw, but use it with caution if this happens. Test it with a control reaction.
- DNA-Lipofectamine 2000 Reagent complexes are stable for at least 5-25 minutes at room temperature. Some users have reported longer stability, up to 6 hours, but this can vary with the dilution media and DNA/RNA samples being used.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Is my lipid (Lipofectin, Lipofectamine, DMRIE-C, Lipofectamine 2000, Oligofectamine, Cellfectin II) supposed to be cloudy?

It is normal to see some turbidity and cloudiness in DMRIE-C, Cellfectin II and Lipofectamine 2000, and the others should be clear. Lipid transfection reagents are sensitive to low temperature; if you put them at temperature below 4 °C they will precipitate or even freeze and lower the efficiency. Most lipids will go cloudy and precipitate upon freezing, and may not be active anymore.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Why do I see precipitates on the cells after transfection?

A small granular-like precipitate may be detected microscopically on the cells after transfection using cationic lipid. This is normal. The presence or absence of this precipitate is not indicative of the transfection efficiency. The precipitate can be caused by presence of excess EDTA or cationic lipid. Use DNA in water or, if in TE, use EDTA concentrations of <0.3 mM in the diluted DNA. Ensure concentrations of cationic lipid reagents do not exceed recommended amounts in complex formation.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How can I improve transfection efficiency with cationic lipids?

1. Select the cationic lipid reagent that is likely to result in highest transfection efficiency for your cell type. Please refer to the Transfection Reagent Selection Guide (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html) to make the right choice.
2. Optimize the cationic lipid reagent and DNA amounts. The most important parameter after the condition of the cells is the ratio of lipid to DNA.
3. Do not use serum during complex formation. Serum may contain components that could interfere with complex formation. We recommend using Opti-MEM I Reduced-Serum Medium for optimal complex formation. However, serum-free DMEM or serum-free RPMI 1640 Medium can be used, keeping in mind that the efficiency of complex formation may not be as high as with Opti-MEM I Reduced-Serum Medium.
4. Do not use antibiotics, EDTA, citrate, phosphate, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the medium used to prepare the DNA-cationic lipid reagent complexes.
5. Cell density should be from 50% to 80% confluency at the time of transfection (for Lipofectamine 2000, we recommend >90% confluency). Cells should be in the mid-log growth phase. For better consistency of results between transfection experiments, it would be best to accurately count your cells with a hemocytometer or with the Countess II FL Automated Cell Counter (Cat. No. AMQAF1000).
6. Make sure the promoter-enhancer of the transfected DNA is compatible with the target cell type.
7. Do not use cationic lipid reagent that has been frozen or stored in a section of the refrigerator where the temperature is below 4 degrees C.
8. Include a positive control for the transfection assay (for example, Cat. No. A14146 for plasmid DNA transfection and Cat. No. 14750100 for siRNA transfection).

Also, please take a look at the tips outlined here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

The protocol sent out with Lipofectamine 2000 indicates to use 24 well plates but I am interested in using 60 mm plates. How do I scale up my transfection with Lipofectamine 2000?

A 60mm plate is 10x greater in surface area than a well from a 24-well plate. Therefore, you would need to scale up all of the components in the reaction by 10x.

The product insert for Lipofectamine 2000 (included with the reagent or can be downloaded from the web site) contains a table indicating the relative surface areas of different cell culture vessels and by what factor needed to scale your transfections.


Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Is Lipofectamine 2000 the same as Lipofectamine?

No. These are two different formulations of lipids and each has been shown to transfect at different efficiencies different cell lines.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Is the passage number of my cells important to consider when doing transfection?

In general, once optimal transfection conditions are determined for a given cell line, it is recommended that cells be passaged less than 20 times to maintain reproducible results. Thus immediately following the determination of optimal conditions, cells should be frozen down so that when the working stock approaches 20 passages, a new batch can be started from the frozen stock.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How does the use of Lipofectamine 2000 reagent improve transfection over the other lipid reagents?

It generally yields better transfection activity (as measured by protein expression) than all other lipids in a majority of the cell lines tested. It includes a streamlined, simple protocol where the complexes are added directly to cells without changing media. This lends itself to high throughput applications. It works very well in the presence or absence of serum. Examples of cells that show the highest transfection efficiency with Lipofectamine 2000 include 293 F, 293 H, BE(2)C, CHO-K1, CHO-S, COS-1, COS7-L, Human Primary Fibroblasts, Ht-29, HT-1080, MDCK, MRC-5, PC12 and Vero.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

I've mistakenly frozen my cationic lipid reagent, can I still use it?

It is safest not to use it, however if the lipid is thawed very slowly at 4°C and its appearance did not change, it is probably worth trying. Lipofectamine 2000 has survived one freeze/thaw without loss of activity.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How do I scale up or down my transfection reaction?

The number of cells, DNA, lipid, and medium volumes should be scaled up proportionately to the surface area of the plate. For commonly used culture vessels, please refer to the information below regarding actual area and area relative to a 24-well plate well.

Vessel type, Area (cm2), Area Relative to 24-well
96-well, 0.3 cm2, 0.2
48-well, 0.7cm2, 0.4
24-well, 2 cm2, 1
12-well, 4 cm2, 2
6-well, 10 cm2, 5
35 mm, 10 cm2, 5
60 mm, 20 cm2, 10
100 mm, 60 cm2, 30
150 mm, 140 cm2, 70
T25, 25 cm2, 12.5
T75, 75 cm2, 37.5
T150, 150 cm2, 75
T162, 162 cm2, 81
T165, 165 cm2, 82.5
40-50 ml, 25 cm2, 12.5
250-300 ml, 75 cm2, 37.5
650-750 ml, 162-175 cm2, 81-87.5
900 ml, 225 cm2, 112.5


Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How do cationic lipids compare with calcium phosphate in transfection efficiency?

For many cell types, higher efficiencies are observed with cationic lipids than with calcium phosphate. Also, cationic lipid data are more reproducible from experiment to experiment. Calcium phosphate is inexpensive however, but pH variation as little as 0.2 can reduce transfection efficiency significantly.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can lipid reagents be used to cotransfect plasmids?

Yes. The standard transfection protocol may be followed by keeping the total amount of DNA in the mixture constant. That is, if your protocol requires 1 ug plasmid, use 0.5 ug of each of two cotransfecting plasmids, or 0.25 ug of each of 4, etc. When performing cotransfections to introduce a selectable marker on a different plasmid, we recommend using a 3:1 to 10:1 molar excess of the plasmid of interest over the selectable plasmid to ensure that the plasmid of interest is present with the selectable plasmid.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the shelf life of the lipid transfection reagents?

Stored at 4°C in a sealed container, the lipids are stable for 12 months. Do not freeze the lipids. At 4°C with long term storage, due to evaporation concentration of lipid may vary, please briefly spin the contents before use. Add less lipid if you start noticing toxicity.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What types of molecules can be transfected with cationic lipid reagents?

Lipid reagents can be used to transfect DNA, RNA, oligonucleotides and siRNA. The DNA can be plasmids, cosmids, or even YAC clones up to 600 kb. Lipofectin and Lipofectamine 2000 Reagent have also been used to transfect cells with proteins (Beta galactosidase and T3 polymerase).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Does the method of generating lipid-DNA complex affect transfection efficiency?

YES. Please follow the recommended procedure for each one of the reagents, found in the product manual.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I use the same amount of any lipid reagent for transfection of my particular cell line?

No. The amount of lipid for each lipid reagent should be optimized for each cell line. Each lipid reagent has different composition/formulation, which will have different impact on each cell line. Therefore please optimize whenever you have a new lipid for each cell line.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

I have optimized my conditions for a particular cell line but I seem to be getting inconsistent results with my transfection. What factors contribute to this inconsistency?

Cells from a different passage number may behave differently. Also, if cells were sitting at confluence prior to plating for transfection, they may not transfect efficiently. To minimize such inconsistencies, passage the cells while they are still growing exponentially. Actively dividing cells transfect better.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What kind of tubes can I use to form DNA:lipid complexes?

Polypropylene, polystyrene, or glass tubes may be used with any of our transfection products.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Are there any guidelines for choosing a lipid transfection reagent?

It is best to optimize for your cells and application. Here are some basic guidelines:

- Lipofectamine LTX and PLUS Reagent: Minimal optimization, excellent efficiency with adherent eukaryotic cells DNA, difficult cell lines
- Lipofectamine Reagent: Adherent eukaryotic cells, DNA, oligonucleotides
- Lipofectin Reagent: Transfecting DNA in eukaryotic cell
- Cellfectin II: Insect cells
- DMRIE-C Reagent: DNA, RNA, suspension cells
- Oligofectamine: Oligonucleotides
- Lipofectamine RNAiMAX: siRNA, pre-miR, miRNA, anti-miR

NOTE: Please also visit our online Transfection Selection Tool to get specific recommendation for your cell line


Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

In the case of the 96-well protocol, Lipofectamine 2000 requires 320 ng DNA, whereas Lipofectamine PLUS, or Lipofectamine LTX requires up to 200 ng DNA. Does this mean that more DNA is needed to transfect efficiently when using Lipofectamine 2000?

Yes, you do need more DNA per well when using Lipofectamine 2000, because the optimal confluency of the cells is 90% (as compared to 50-80% confluent with Lipofectamine, or Lipofectamine LTX. Therefore you need more DNA in order to ensure that the maximum number of cells are transfected. With Lipofectamine 2000, we have found empirically that the higher confluence (~90%) of cells at the start of transfection improves transfection efficiency.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Why is the lot number on the package of the Lipofectamine 2000 Transfection Reagent/product different from the lot number on the Lipofectamine tube’s label?

There is an overall lot number associated with the catalog number of the product. This lot number is the packaging lot number and can be found on the package. The individual components within the package have a component reference number associated with ‘filling lot numbers’. The filling lot numbers are used for internal traceability purposes.

The shipping for my Lipofectamine 2000 Transfection Reagent was delayed. Can I still use it?

Yes, Lipofectamine 2000 Transfection Reagent can be shipped at room temperature without impacting the performance. Please see this white paper (also found in the Documents section on the Lipofectamine 2000 product page) on this topic.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How long after transfection should I harvest RNA when using Lipofectamine 2000 Transfection Reagent?

The time of RNA harvest can vary from 24-72 hrs after transfection. We recommend conducting a pilot experiment with a time course. Protein harvest should follow the same timeline.

I accidentally froze my Lipofectamine 2000 Transfection Reagent. Can I still use it?

We do not recommend using Lipofectamine 2000 Transfection Reagent that has been frozen. Once frozen, the reagent will no longer perform as intended.