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View additional product information for pcDNA™3.4 TOPO™ TA Cloning Kit - FAQs (A14697)
128 product FAQs found
我们不建议将感受态细胞保存在液氮中，因为极端温度会损害细胞。另外，装感受态细胞的塑料管子可能承受不了如此低的温度，从而发生破裂。
我们推荐将感受态细胞存储在-80摄氏度。高于这个温度，即使存储时间很短，也会显著降低其转化效率。
大多数293细胞系均能适应从传统含血清或其它无血清培养基向Expi293表达培养基中的切换按照手册中所描述的直接切换法或序贯切换法来操作，大多数293细胞系均能适应从传统含血清或其它无血清培养基向Expi293表达培养基中的切换。 注意：切换后的细胞可能无法达到与Expi293F细胞同等的高水平表达。
pcDNA 3.4-TOPO TA载体在pcDNA 3.3-TOPO TA载体上进行了一个重要改进。它包含了能够使蛋白水平相比pcDNA 3.3-TOPO TA载体提升2-3倍的WPRE元件（旱獭转录后调控元件）。
我们推荐使用pcDNA 3.4-TOPO TA载体（货号A14697）。这一载体含有天然的全长CMV启动子和克隆位点下游的WPRE（旱獭转录后调控元件），这两个元件均有利于基因的高水平表达（表达比pcDNA3.3 TOPO载体高大约2-3倍，而后者又比标准的pcDNA载体高2-5倍）。当然，实际表达水平还要视具体蛋白而定。
除了Opti-MEM I低血清培养基为无血清但不是非动物源性的外，本系统中的其余成份均为非动物源性的，请参考应用说明(https://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.5943.file.dat/expi-293-animal-origin-free-co25751.pdf): 在非动物源性的条件下使用Expi293表达系统。
我们为蛋白的稳定高产量生产提供Freedom CHO-S与Freedom DG44试剂盒。我们为瞬时高产量生产提供ExpiCHO表达系统，Expi293表达系统，FreeStyle 293 表达系统，FreeStyle Max 293表达系统和FreeStyle Max CHO表达系统。如需在Exp293F细胞中高效表达功能性膜蛋白，我们还提供了Expi293 MembranePro 表达系统（货号A25869，A25870），这款产品将Expi293的扩展性/易用性与MembranePro技术整合起来，能够将膜蛋白的表达产量大大提升，超出标准贴壁培养的MembranePro功能性蛋白表达系统的表达产量20倍以上。请参见 应用说明(https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/ProteinExpressionAnalysis/pdfs/PG1391-PJ6257-CO28704-Expi293Adaption-MembraneProTech_AppNoteGlobal-FHR.pdf) 以了解更多详情。
您可能需要尝试不同的插入片段:载体比例，范围从1:1至15:1。
公式：
length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 insert:vector ratio
(插入片段长度 (bp) X 载体重量(ng) ) / 载体长度 (bp) = 插入片段:载体比例为1:1时所需的插入片段重量(ng)
是的，该酶混合物会使一部分PCR产物留下3′端 A尾。然而相对于单独采用Taq聚合酶，克隆效率会大幅度下降。建议在PCR产物添加3′端 A尾后再进行TA克隆。
仅使用载体进行转化不是推荐的阴性对照实验。拓扑异构酶改构过程不是一个效率100%的过程，因此，在您的混合物中将会存在“不含插入片段的载体”，因此会产生克隆。
磷酸化的产物可以进行TA克隆但不能进行TOPO克隆。这是因为所需的磷酸基团已经包含在载体DNA和拓扑异构酶形成的中间体复合物中。TOPO载体含有一个与拓扑异构酶共价结合的3' 磷酸基团和一个5'磷酸基团。非TOPO载体的线性载体（TA和Blunt）含有一个3' OH基团和一个5'磷酸基团。磷酸化产物在进行TOPO-克隆之前应该用磷酸酶（CIP）处理。
你可能克隆了一段假阳性序列。TA和TOPO克隆对于存在于某些PCR反应中的小片段（< 100 bp）非常高效。使用硅基DNA纯化系统对插入片段进行凝胶纯化或者电洗脱。确保所有溶液均不含核酸酶（例如避免共用溴化乙锭染色器皿）。
出现这一问题的原因可能有：
•插入片段没有破坏lacZ基因的读码框。如果你的插入序列较短（< 500 bp），则可能出现浅蓝色克隆。可以分析一些这类克隆，因为它们可能含有插入片段。
•使用了不能在3´ 末端加A的聚合酶。如果您使用的是具有校读活性的聚合酶，例如AccuPrime Pfx 或Platinum Pfx，您将需要进行一个后续Taq酶处理步骤以添加3’ A突出末端。
• PCR产物在连接之前进行了凝胶纯化。凝胶纯化可以去除单个的3’ A突出碱基。如果没有进行凝胶纯化，那么请优化您的PCR反应以便可以直接从PCR产物进入克隆步骤。
• PCR产物在进行连接反应前被存放了很长时间。请使用新鲜PCR产物。即使仅存放1天，连接效率也会下降。
•连接反应中加入了太多扩增反应的成分。PCR反应体系内的高盐成分会抑制连接反应。在连接反应中应使用不超过2-3 μl的PCR反应混合物。
•连接反应中的载体：插入片段摩尔比可能不正确。估计PCR产物的浓度。使连接反应中载体和插入片段的摩尔比大约为1:1或1:3。
通常平板上应该有少量不含插入片段的白色克隆。这些克隆通常比其它克隆大一些，是由于罕见的重组事件会导致质粒上部分序列缺失引起的（通常是从多克隆接头到F1起始点附近的一个位点）。为找到含有插入片段的克隆，最好挑选各种颜色和形态的克隆进行分析。同一插入片段经常会由于插入方向的不同而产生两种完全不同的克隆形态。
无克隆产生的原因可能是出现了下列问题：
•细菌不是感受态细胞。使用包含在One Shot模块内的pUC18载体检查感受态细胞的转化效率。
•平板的抗生素浓度不正确，或者平板过于陈旧。使用100 μg/mL的氨苄青霉素或50 µg/mL的卡那霉素。确保氨苄平板是新鲜的（储存时间小于1个月）。
•产物被磷酸化了（仅针对TOPO克隆而言）。磷酸化的产物可以进行TA-克隆但不能进行TOPO-克隆。这是因为连接所需的磷酸基团已经包含在载体DNA和拓扑异构酶-形成的中间体复合物中了。TOPO载体含有一个与拓扑异构酶共价结合的3' 磷酸基团和一个5'磷酸基团。非TOPO载体（TA和Blunt）含有一个3' OH基团和一个5'磷酸基团。磷酸化产物在进行TOPO-克隆之前应该用磷酸酶（CIP）处理。
请考虑以下可能原因：
• pH > 9：检查PCR扩增反应的pH值，并使用1 M Tris-HCl, pH 8的缓冲液进行调节。
• PCR产物过多（或过度稀释）：减少PCR产物的用量（或增加其浓度)
• PCR反应延伸不完全：确保PCR反应最后包含一个7到30分钟的延伸步骤。长的PCR产物将需要更长的延伸时间。
•克隆长插入片段（>1 kb）：请尝试以下一个或全部建议：提高插入片段用量。延长 TOPO克隆反应孵育时间。使用硅基DNA纯化系统（如PureLink系统）或者电洗脱法对插入片段进行凝胶纯化。确保所有溶液均不含核酸酶（例如避免共用溴化乙锭染色器皿）。
•尽管你使用了Taq聚合酶但是PCR产物仍然没有足够的3´ A突出：提高最后的延伸时间以确保所有的3´末端被腺苷化。Taq聚合酶在模板链的A后面再添加一个A时的效率较低。Taq聚合酶在模板链的C后边添加一个A时的效率最高。您可能需要重新设计引物以使它们包含一个5´ G而非一个5´ T。
请尝试以下建议以提高克隆数量。
•向反应中加入6X盐溶液后，提高TOPO反应在室温孵育的时间。
•电转化能显著提高克隆数量；通常能提高10到20倍。但是，如果进行电转化，重要的一点是TOPO反应混合物要包含稀释的盐溶液，或者，为获得最佳结果，在转化前进行沉淀。为获得高电转化效率，建议如下操作： ◦向 6 µL TOPO反应体系中加入100 µL双蒸水并在37度再孵育10分钟。
◦加入10 µL 3 M 醋酸钠，2 µL 20 µg/µL糖原，300 µL 100%乙醇进行沉淀。置于干冰上或–80摄氏度20分钟，4摄氏度最高速离心15分钟。使用800 µL 80%乙醇洗涤沉淀团块，最高速离心10分钟，倒去乙醇，离心1分钟，然后移除剩余乙醇，避免碰到沉淀团块。干燥沉淀团块（空气干燥或真空吸干）。
◦将沉淀重悬于10 µL ddH2O中并根据常用电转步骤使用3.3 µL 重悬的DNA进行电转化。该电转化将产生比使用同样的TOPO-反应体系进行化学转化时多出高达20倍的克隆。加入100 µL ddH2O并孵育10分钟并不是绝对必要，但是它能充分的稀释反应并可能有助于拓扑异构酶的失活，以使得电转化更容易进行。
我们建议您使用我们的TOPO TA cloning kit for sequencing，它包含pCR4 TOPO载体，或者使用我们的Zero Blunt TOPO PCR cloning kit for sequencing,，它包含pCR4Blunt-TOPO载体。
因为您的PCR产物较小，我们建议您使用pCR2.1 TOPO载体。这一大小的片段将不能完全破坏pCR4-TOPO载体内的ccdB基因，因此你可能不能得到克隆，因为ccdB对E. Coli是致死的。
常规 TOPO TA克隆试剂盒对于2-3 kb左右的PCR产物能进行高效克隆。当PCR产物大于3kb时，克隆效率明显下降。TOPO XL PCR克隆试剂盒已优化可用于长(3- 10kb) PCR产物的TOPO克隆。如果使用常规TOPO试剂盒，这里是一些提高效率的建议：
1.使用结晶紫替代溴乙锭（EtBr）在凝胶分离过程中对PCR产物染色以避免DNA切口的出现。
2.提高TOPO反应孵育时间至30分钟。
3.保持较低的插入片段：载体摩尔比，最佳比值为1:1。
4.将反应稀释至20 µl，同时保持相同数量的载体和插入片段。将盐溶液体积增加到3.7 µl以补偿增加的体积。稀释反应可以降低对于载体末端的竞争。
将TOPO载体和插入片段反应混合物在4°C保存一星期被证明不会影响 TOPO反应的克隆效率，因为仍有超过95%的克隆含有插入片段。但是，总克隆数降低了10倍。将TOPO反应混合物放于4°C过夜后总克隆数和新鲜TOPO反应混合物比较没有或仅有轻微降低。
6X 终止溶液的成分是0.3 M NaCl，0.06 M MgCl2，而6X盐溶液的成分是1.2 M NaCl，0.06 M MgCl2。终止溶液仅包含在 TOPO XL 克隆试剂盒，而盐溶液目前包含在所有其它TOPO试剂盒中。这些溶液可以阻止拓扑异构酶重新结合并使质粒产生切口，后者将降低TOPO反应的克隆数量。
在进行一个TOPO克隆反应时，2 µl的PCR反应可包含最多10% DMSO或1.3 M甜菜碱而不影响TOPO反应。甲酰胺和高水平的甘油将抑制反应。这些试剂通常会被加入到PCR反应中以提高PCR产物的产量，例如，用来降低二级结构的影响或帮助扩增高GC含量的序列。我们还没有测试甘氨酸或乙酰胺对TOPO克隆反应的影响。
在使用任何TOPO载体进行克隆时PCR引物都不能有5´磷酸基团，因为5´磷酸基团抑制TOPO克隆反应。磷酸化的产物可以进行TA克隆但不能进行TOPO克隆。这是因为所需的磷酸基团已经包含在载体的拓扑异构酶-DNA中间体复合物中。TOPO载体含有一个与拓扑异构酶共价结合的3' 磷酸基团和一个5'磷酸基团。非TOPO线性载体（TA和Blunt）含有一个3' OH基团和一个5'磷酸基团。磷酸化产物在进行TOPO-克隆之前应该用磷酸酶（CIAP）处理。使用CIAP处理可将效率提高至25%。使用5'-生物素标记的引物（或任何5'-标记，包括 5’-Cy5标记）生成的PCR产物都因为空间位阻而不能连接到任何TOPO载体中。
如果凝胶电泳显示PCR产物是干净的，没有可见的非特异性条带或引物二聚体，那么凝胶纯化是不需要的。如果PCR产物大于1.5 kb或如果凝胶电泳显示可见的非特异性条带或引物二聚体，则建议进行凝胶纯化。小片段产物比大片段产物克隆进载体的效率高很多，所以在克隆之前应该将它们去除。在PCR产物凝胶纯化后会损失一些A突起，以及一些PCR产物的损失，这些因素会导致总克隆数的轻微减少。但是，带有插入片段的克隆的比例不会发生变化，该比例通常大于90%。
我们建议使用新鲜PCR产物用于TOPO克隆反应。
TA克隆使用T4连接酶连接插入片段和载体，而TOPO TA克隆使用拓扑异构酶的本身性质，其能够在室温孵育5分钟内连接插入片段和载体。TOPO TA克隆重组率超过95%，而TA克隆重组率大于80%。
我们提供一项TOPO克隆载体改造服务。我们的科学家可以将您的载体改造为适用于blunt TOPO 克隆, TOPO TA 克隆, 或PCR产物定向TOPO 克隆。
可以，我们的pCR2.1 TOPO TA (货号450641), pCR4-TOPO TA (货号450030), pCRBluntII-TOPO (货号450245)均可提供单独的不含感受态的克隆试剂盒。
我们不建议这么做，因为这些载体的末端结合有拓扑异构酶DNA蛋白复合物。
我们建议起始摩尔比为1:1(插入片段：载体)，范围为0.5：1到2：1(插入片段：载体)。TOPO克隆反应中，2kb PCR产物的ng值应在5- 10ng之间。
请在TOPO克隆前考虑以下问题：
• TOPO克隆不能连接带有5’磷酸基团的DNA。
• TOPO克隆效率会随着插入片段长度增加而降低，超过3 kb 建议使用TOPO XL克隆试剂盒。
• TOPO载体包含不同的抗生素抗性标记，这一点应该在购买之前考虑。
• TOPOTA载体可连接带有3’ A突出末端的片段，而Zero Blunt载体可连接带有平末端的片段。
TOPO及TOPO TA载体（非定向）加入了酚红染料。室温下溶液应该为粉红色（或者黄色）。如果在加入PCR产物后溶液变为蓝色，说明PCR缓冲液太偏碱性，转化的克隆数会变少。当溶液为黄色时，表明pH值为酸性。在pH 2.0条件下，TOPO载体仍能保持较高克隆效率。定向的TOPO及Zero Blunt TOPO载体加入了溴酚蓝。
亚克隆级别的DH5α可以使用，但是转化的效率会低一些（10e6 vs 10e9），因此得到的克隆数可能较少。 Top10F’细胞也可以使用，但是如果要进行蓝白斑筛选，需要同时加入IPTG和X-gal，因为带有F’游离体的细胞会表达lacIq抑制子。
确保您使用了正确的抗生素和合适的浓度。另外，确保抗生素没有过期。如果克隆的形态和预期不同，那么污染可能是一个原因。检测您的SOC培液和LB培液。
以下是一些建议：
•小片段/接头而不是您的插入片段被克隆进了您的载体。要解决这一问题，请在连接前对插入片段进行凝胶纯化。
•确保使用了正确的X-gal和/或IPTG（当载体带有lacIq标志物时）浓度。
•在平板上涂布X-gal和/或IPTG时，留出足够的时间让试剂充分扩散进入平板。
•孵育平板足够长的时间以确保颜色完全显现。
虽然没有特定菌株更适合进行大质粒转化，但是关注转化效率是非常重要的。对于大质粒，推荐使用效率最高的化学感受态细胞，例如OmniMAX 2，TOP10，等等。对于超过20 kb的质粒，建议使用电转感受态细胞以获得最高的转化效率。
无论使用何种菌株，您所要做的第一件事都是降低生长温度至30摄氏度或更低（室温）。慢速生长通常可以让E. coli更加耐受复杂序列。如果降低生长温度没有帮助，则您可能需要考虑使用其它感受态细胞比如Stbl2或Stbl4细胞，这类细胞已被证明在同等条件下对于上述复杂序列的耐受程度比其它菌株高。
我们建议尝试以下措施：
•进行pUC19转化对照；这可以告诉您所用感受态细胞的转化能力信息。
•检查平板的过期时间以及是否使用了正确的培液（LB/琼脂）。
•确认使用了正确的抗生素并且浓度也是对的。
TOP10F’中的F’游离体带有一个lacIq标志物，它可以过表达lac抑制子。必须将IPTG和X-gal一起加入平板中才能在该菌株内看到β-半乳糖苷酶的表达和蓝色的出现。而TOP10则不需要IPTG就能进行蓝白斑筛选。
以下这些因素会导致这一情况：涂板时所用的SOC培液或其它培液受到了污染，DNA被具有氨苄抗性的微生物所污染，平板时间过久氨苄降解失效，或者感受态细胞本身被污染了。
如果您使用的是一种mcr/mrr(+)感受态细胞株，其细胞内的酶可能会识别酵母基因组DNA上的真核细胞甲基化序列并将其删除或重排。请尝试用一种mcr/mr(–)菌株例如Top10, DH10B, 或OmniMAX 2进行转化。
XbaI切割位点是一个Dam甲基化敏感限制性酶切位点。TOP10是一种dam(+)菌株，它表达甲基化酶Dam。您可以试试重新转化一种dam(–)菌株，例如INV110。其它dam(–) (以及dcm(–))敏感的限制性酶切位点包括：
Dam: Bcl I, Cla I, Hph I, Mbo I, Mbo II, Taq I, Xba I, BspH I, Nde II, Nru I
•Dcm: Ava II, EcoO 109 I, EcoR II, Sau96 I, ScrF, Stu I, Aat I, Apa I, Bal I, Kpn I, ISfi I
1.使用pUC或基于pUC的载体，这些载体含有lacZ基因的一部分，可以进行α补偿。
2.选择一种带有 lacZdeltaM15标记的E. Coli菌株。
3.将转化混合液涂布在含有X-gal的平板上。在一个100 mm平板上涂布50 µg的2% X-gal或者100微升2% bluo-gal （均可溶解于DMF或50:50的DMSO：水混合物中）并晾干。此外，也可在倒板前直接向冷却后的培液中加入终浓度50 µg/mL的X-gal或300 µg/mL的bluo-gal。平板在4摄氏度可稳定保存4周。
4.如果菌株带有lacIq标志，则需加入IPTG以诱导lac启动子。在100 mm平板上涂布30 µl的100 mM IPTG（溶于水中）。此外，也可以在倒板前直接向冷却后（大约59度）的培养液中加入IPTG至终浓度1 mM。平板在4摄氏度可稳定保存4周。
5.如果要使用 X-gal或bluo-gal进行蓝白斑筛选，那么不要将E. coli 涂布在含有葡萄糖的培养基上。因为葡萄糖会作为底物和X-gal或bluo-gal竞争从而阻止细胞变蓝。
对于长期存储，建议制作甘油存储液并保存在-70摄氏度。请按以下步骤操作：
1.挑取1个克隆到5 mL的LB培液或SOC培液中。37摄氏度培养过夜。
2.制备甘油溶液：6 mL的SOB培液加4 mL甘油。
3.细胞与甘油溶液等体积混合并混匀。
4.在乙醇/干冰混合物中冷冻然后存储在-70摄氏度。
是的，这是可以的。我们推荐使用饱和量的DNA（每种质粒10 ng）。确保不同质粒的复制起始点不同，以便它们可以同时存在于细胞中。如果复制起始点相同，则两种质粒会互相竞争，其中一种质粒即使稍有劣势也可能会丢失。另外，带有一个质粒的细胞可以通过电转导入第二个质粒，而不会破坏已有质粒。
在平板上，我们建议50 µg/mL X-gal和1 mM IPTG (0.24 mg/mL)。当直接涂布到琼脂平板上时，我们建议在琼脂平板表面使用40–50 µl溶于DMF的40 mg/mL 的X-gal (2% 储液)和30–40 µl的100 mM IPTG。让X-gal和IPTG向琼脂内进行扩散大约1小时。不要在含有葡萄糖的培养基上涂布，因为葡萄糖会和X-gal或bluo-gal竞争从而阻止细胞变蓝。
感受态细胞效率通过测定转化效率得到。转化效率等于每µg质粒DNA形成的转化子的数量，或者克隆数量(cfu/ µg)。
下面是一些能帮助您获得最高转化效率的建议：
•将感受态细胞在冰浴中解冻而不是在室温中解冻；不要对细胞进行涡旋震荡。
•感受态细胞解冻后，立刻在其中加入DNA。
•确保孵育时间是符合感受态细胞实验方案中针对该菌种所列出的要求的；改变孵育时间会降低效率。
•去除您的DNA样本中的盐分及其它污染；转化前可以使用离心柱或苯酚/氯仿抽提、乙醇沉淀的方法纯化DNA。
DH5α细胞是常规克隆的常用菌株，但它是mcr/mrr+的，因此不推荐用于基因组克隆。另一方面，TOP10感受态细胞包含突变的mcr/mrr，因此是常规克隆不错的选择而且可以用于甲基化的DNA的克隆，如真核基因组DNA。我们的Mach1菌株是生长最迅速的具有T1噬菌体抗性的菌株。
这些小的克隆很有可能是由于氨苄活性降低产生的。这些克隆是生长在氨苄活性降低的LB培养板上的未转化细胞。为了避免这种情况，你可以尝试：
1.降低细胞铺板密度。
2.使用新鲜的LB氨苄培养板或者用羧苄青霉素代替氨苄青霉素。
3.培养板不能在37摄氏度下孵育超过20小时。β-内酰胺酶是一种由氨苄抗性基因产生的酶，它由具氨苄抗性的转化菌分泌，能够使转化的菌落周围的抗生素失活。由于筛选试剂的失活而使得卫星菌落（不是真正的氨苄抗性）能够生长。如果使用羧苄青霉素也会产生同样情况。
其中一个原因是可能与插入片段的毒性有关。这种毒性不能影响在固体培养基上缓慢生长的细胞，但是在快速生长条件（如液体培养基中）下，其毒性更强。建议：
使用TOP10F’或其他带有LacIq阻抑物的菌种。
2.尝试使用任何其它的适用于克隆的菌株。
3.将培养温度降至27至30摄氏度并延长培养时间。
4.不生长的另一可能的原因是噬菌体污染。此种情况下，我们推荐使用具有T1噬菌体抗性的菌株，如DH5α-T1R。
这可能是由于插入DNA在TOP10大肠杆菌中不稳定造成的。此种情况下，Stbl2、Stbl4等大肠杆菌菌种对于有正向重复的DNA、反转录病毒序列、及高GC含量的DNA扩增的效果优于其它菌种。
一些可能的原因及补救措施如下：
•连接酶活性低。检查连接酶生产日期以及缓冲液的功能。
•感受态细胞没有转化能力。使用超螺旋载体，如puc19，检测感受态细胞效率。
•两个片段都已去磷酸化。
•限制性内切酶及残留的缓冲液抑制连接反应。不妨尝试转化未切割载体，使用苯酚去除内切酶，或者连接前进行PCR产物纯化／胶回收。
•抗生素使用错误。检查质粒及培养板并确保使用了正确浓度的抗生素。
如果以上均不适用，转化后克隆数低或没有克隆可能是由于DNA插入片段在感受态细胞中不稳定造成的。此种情况下，Stbl2、Stbl3、或Stbl4等大肠杆菌菌种对于有多片段重复的DNA、反转录病毒序列、及高GC含量的DNA扩增的效果优于其它菌种。
如果使用包含lac启动子和LacZ α片段（用于α互补）的载体，蓝/白筛选可作为筛选插入片段是否存在的工具。X-gal作为LacZ酶的底物需要加到反应板中，是蓝/白斑筛选必需的。完全破坏LacZ基因表达所需的最小插入片段大小为400 bp。如果存在Laclq抑制子（来自宿主细胞，如TOP10F'，或由质粒表达），它将抑制lac启动子介导的表达，从而阻碍蓝/白筛选。因此，当存在Laclq抑制子时，必须使用IPTG抑制Laclq。只有抑制了Laclq，才能实现蓝/白筛选中的lac启动子介导的表达。
含有LacZ-ccdB表达框的TOPO载体，可通过破坏对大肠杆菌致死的基因ccdB的表达而直接筛选重组体。PCR产物的连接，可破坏LacZ-ccdB融合基因的表达，从而仅允许转化所得的阳性重组体生长，而含有非重组载体的细胞则将死亡。因此，无需蓝/白筛选。对含有LacZ-ccdB表达框的TOPO载体的克隆进行蓝/白筛选时，可看见菌落呈现不同的蓝色阴影。根据我们的经验，浅蓝色和白色的菌落通常含有插入片段。浅蓝色克隆的形成是由于在有插入片段的情况下发生了部分LacZ基因的转录但ccdB的表达没有达到杀死细胞的水平，这是和插入片段有关的。为了完全破坏lacZ基因的表达，插入片段必须>400 bp；因此，一个300 bp的插入片段可产生浅蓝色菌落。出现不含插入片段的白色菌落，通常是因为ccdB基因的自发性突变。
为了确保破坏ccdB基因表达，防止细胞死亡，需要的插入片段最小为150 bp。（参考文献：Bernard et al., 1994. Positive-selection vectors using the F plasmid ccdB killer gene. Gene 148: 71-74.）
含F质粒的菌株，如TOP10F’，不推荐用于转化和筛选任何含ccdB基因的TOPO载体的重组克隆。F质粒可编码ccdA蛋白，一种ccdB旋转酶-毒素蛋白的抑制剂。ccdB基因也存在于F质粒的ccd（细胞死亡控制）位点。该位点含2个基因，ccdA和ccdB，可分别编码含72和101个氨基酸的蛋白。ccd位点通过杀死分裂后不含F质粒的细胞而维持F质粒的稳定性的。当ccdA蛋白不发挥抑制作用时，ccdB蛋白是一种强力的可以杀死细胞的蛋白。
如果使用包含lac启动子和LacZ α片段（用于α互补）的载体，蓝/白筛选可作为筛选插入片段是否存在的工具。X-gal作为LacZ酶的底物需要加到反应板中，是蓝/白斑筛选必需的。完全破坏LacZ基因表达所需的最小插入片段大小为400 bp。如果存在Laclq抑制子（来自宿主细胞，如TOP10F'，或由质粒表达），它将抑制lac启动子介导的表达，从而阻碍蓝/白筛选。因此，当存在Laclq抑制子时，必须使用IPTG抑制Laclq。只有抑制了Laclq，才能实现蓝/白筛选中的lac启动子介导的表达。X-gal（也称为5-溴-4氯-3-吲哚-β-D-半乳糖苷）可溶于DMSO或DMF，其溶液形式可在冰箱中储存长达6个月。溶液需避光保存。X-gal和IPTG在琼脂板中的终浓度：倾注平板前，在培养基中加入X-gal至终浓度20 mg/mL，IPTG 加至终浓度0.1 mM。若直接涂在反应板表面，则加入40 µl X-gal (20 mg/mL储液)和4 µl IPTG (200 mg/mL储液)。
不可以，这些载体不包含功能性启动子以表达您的目的基因。这些载体通常用于亚克隆或测序。
TA克隆
这种克隆方法最初是为配合纯Taq聚合酶（天然的、重组的、热启动）使用而设计的；然而，某些高保真Taq酶和Taq酶混合物通常也适合TA克隆。即使Taq与具有校正能力的聚合酶以10:1或15:1的比例，仍可以产生足够的3’ A突出端去做TA克隆。
推荐使用的我公司的聚合酶包括Platinum Taq、Accuprime Taq、Platinum或Accuprime Taq High Fidelity、AmpliTaq、AmpliTaq Gold或AmpliTaq Gold 360等。
平末端克隆
使用Platinum SuperFi DNA聚合酶等具有校对能力的酶。
定向TOPO克隆
Platinum SuperFi DNA聚合酶效果良好。
如果你想使用包含Taq聚合酶和一个校对活性聚合酶的DNA聚合酶混合物，Taq酶和校对活性聚合酶的比例必须超过10:1以确保PCR产物带有3´ A突起。如果你使用的聚合酶混合物没有足够的Taq聚合酶或仅含有校对活性聚合酶，你可以在PCR反应后添加3´ A突起。请参阅产品手册获取细节。 适合TOPO TA克隆的一些Taq酶混合物的例子包括Platinum Taq DNA Polymerase High Fidelity和AccuPrime Taq DNA Polymerase High Fidelity。
我们建议使用 pcDNA3.4 TOPO TA 载体 (货号：A14697)，以获得最佳表达效果。该载体包含全长 CMV 启动子，在克隆位点下游有一个土拨鼠转录后调控元件 (WPRE)。但是，研究发现其他以 CMV 为基础的载体表达效果与 pcDNA3.4 TOPOTA 载体类似，因此任何以 CMV 为基础的载体都是ExpiCHO系统的理想选择。
We do not recommend storing competent E. coli strains in liquid nitrogen as the extreme temperature can be harmful to the cells. Also, the plastic storage vials are not intended to withstand the extreme temperature and may crack or break.
We recommend storing our competent E. coli strains at -80°C. Storage at warmer temperatures, even for a brief period of time, will significantly decrease transformation efficiency.
Other 293 cell lines may be used with the Expi293 Expression System. However, before these cell lines may be used for transfection studies, they must be adapted to serum-free, suspension culture in Expi293 Expression Medium and evaluated for transfection and expression.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
pcDNA 3.4-TOPO TA vector is an improvement over pcDNA 3.3-TOPO TA vector. It contains the WPRE (Woodchuck Posttranscriptional Regulatory Element) that allows for 2- to 3-fold higher levels of expression than pcDNA 3.3-TOPO TA vector.
We recommend using the pcDNA 3.4-TOPO TA vector (Cat. No. A14697). This vector contains the native, full-length CMV promoter and a WPRE (Woodchuck Posttranscriptional Regulatory Element) downstream of the cloning site, both of which contribute to high level gene expression (about 2-3 fold higher expression than with pcDNA3.3 TOPO vector, which in turn provides 2-5 fold higher expression than with a standard pcDNA vector). Of course, the expression level is also protein-dependent.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
All the components of the system are animal-origin free except for the Opti-MEM I Reduced Serum Medium that is serum-free but not animal-origin free. Please see the Application Note for using the Expi293 Expression System under animal origin-free conditions:
http://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.5943.file.dat/expi-293-animal-origin-free-co25751.pdf
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
For stable high-yield expression, we offer the Freedom CHO-S Kit (Cat. No. A1369601) and Freedom DG44 Kit (Cat. No. A1373701). For transient high-yield expression, we offer the ExpiCHO Expression System (Cat. No. A29133), Expi293 Expression System (Cat. No. A14635), FreeStyle 293 Expression System (Cat. No. K900001), FreeStyle Max 293 Expression System (Cat. No. K900010), and FreeStyle Max CHO Expression System (Cat. No. K900020). For high-yield expression of functional membrane proteins in Exp293F cells, we offer the Expi293 MembranePro Expression System (Cat. Nos. A25869, A25870) that combines the scalability and ease of use of Expi293 and the technology of MembranePro to allow an increase of more than 20-fold in membrane protein yield compared to the standard, adherent culture MembranePro Functional Protein Expression System. Please see the Application Note (http://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/ProteinExpressionAnalysis/pdfs/PG1391-PJ6257-CO28704-Expi293Adaption-MembraneProTech_AppNoteGlobal-FHR.pdf) for more details.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The optimal ratio is 1:1 insert to vector. Optimization can be done using a ratio of 0.5-2 molecules of insert for every molecule of the vector.
Equation:
length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 insert:vector ratio
Yes, the enzyme mix leaves 3' A-overhangs on a portion of the PCR products. However, the cloning efficiency is greatly decreased compared to that obtained with Taq polymerase alone. It is recommended to add 3' A-overhangs to the product for TA cloning.
Using the vector only for transformation is not a recommended negative control. The process of TOPO-adaptation is not a 100% process, therefore, there will be “vector only” present in your mix, and colonies will be obtained.
Phosphorylated products can be TA cloned but not TOPO cloned. This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector. TOPO vectors have a 3' phosphate to which topoisomerase is covalently bound and a 5' phosphate. Non-TOPO linear vectors (TA and Blunt) have a 3' OH and a 5' phosphate. Phosphorylated products should be phosphatased (CIP) before TOPO cloning.
You may be cloning in an artifact. TA and TOPO Cloning are very efficient for small fragments (< 100 bp) present in certain PCR reactions. Gel-purify your PCR product using either a silica-based DNA purification system or electroelution. Be sure that all solutions are free of nucleases (avoid communal ethidium bromide baths, for example.)
There could be a few possibilities for this:
- The insert does not interrupt the reading frame of the lacZ gene. If you have a small insert (< 500 bp), you may have light blue colonies. Analyze some of these blue colonies as they may contain insert.
- A polymerase that does not add 3' A-overhangs was used. If you used a proofreading enzyme, you will need to do a post-reaction treatment with Taq polymerase to add the 3' A-overhangs.
- PCR products were gel-purified before ligation. Gel purification can remove the single 3' A- overhangs. Otherwise, optimization of your PCR can be performed so that you can go directly from PCR to cloning.
- The PCR products were stored for a long period of time before ligation reaction. Use fresh PCR products. Efficiencies are reduced after as little as 1 day of storage.
- Too much of the amplification reaction was added to the ligation. The high salt content of PCR can inhibit ligation. Use no more than 2-3 µl of the PCR mixture in the ligation reaction.
- The molar ratio of vector:insert in the ligation reaction may be incorrect. Estimate the concentration of the PCR product. Set up the ligation reaction with a 1:1 or 1:3 vector:insert molar ratio.
On a typical plate there are a few white colonies which do not contain insert. These are usually larger than the other colonies and are due to a deletion of a portion of the plasmid sequence by a rare recombination event (usually from the polylinker to a site in the F1 origin). To find a colony with an insert it is best to pick clones of a variety of color and pattern for analysis. Often an insert will generate two distinct patterns according to its orientation.
No colonies may occur due to the following problems:
Bacteria were not competent. Use the pUC18 vector included with the One Shot module to check the transformation efficiency of the cells.
- Incorrect concentration of antibiotic on plates, or the plates are too old. Use 100 µg/mL of ampicillin or 50 µg/mL kanamycin. Be sure ampicillin plates are fresh (< 1 month old).
- The product was phosphorylated (TOPO cloning only). Phosphorylated products can be TA-cloned but not TOPO-cloned. This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector. The TOPO vector has a 3' phosphate to which topoisomerase is covalently bound and a 5' phosphate. The non- TOPO vectors (TA and Blunt) have a 3' OH and a 5' phosphate. Phosphorylated products should be phosphatased (CIP) before TOPO-cloning.
Please consider the following possible causes:
- pH > 9: Check the pH of the PCR amplification reaction and adjust with 1 M Tris-HCl, pH 8.
- Excess (or overly dilute) PCR product: Reduce (or concentrate) the amount of PCR product.
- Incomplete extension during PCR: Be sure to include a final extension step of 7 to 30 minutes during PCR. Longer PCR products will need a longer extension time.
- Cloning large inserts (>1 kb): Try one or all of the following suggestions: Increase amount of insert. Incubate the TOPO cloning reaction longer. Gel-purify the insert using either a silica-based DNA purification system (e.g., PureLink system) or electroelution. Be sure that all solutions are free of nucleases (avoid communal ethidium bromide baths, for example.)
- PCR product does not contain sufficient 3' A-overhangs even though you used Taq polymerase: Increase the final extension time to ensure all 3' ends are adenylated. Taq polymerase is less efficient at adding a nontemplate 3' A next to another A. Taq is most efficient at adding a nontemplate 3' A next to a C. You may have to redesign your primers so that they contain a 5' G instead of a 5´ T.
Please try the suggestions below to increase the number of colonies.
- Longer incubation of the TOPO cloning reaction at room temperature, provided that the 6X Salt solution is added to the reaction.
- Electroporation can give significant increases in colony numbers; often 10-20 fold higher. However, if doing electroporation, it is important that the TOPO reaction mix contains diluted Salt solution or, for best results, precipitated prior to transformation. For high primary transformants by electroporation it is recommended to:
- Add 100 µL double diH2O to the 6 µL TOPO reaction and incubate 10 more minutes at 37 degrees C.
- Precipitate by adding 10 µL 3 M Na-Acetate, 2 µL 20 µg/µL glycogen, 300 µL 100% ethanol. Place on dry ice or –80 degrees C for 20 min, spin at top speed in a microcentrifuge at 4 degrees C for 15 min. Wash pellet with 800 µL 80% ethanol, spin at top speed for 10 min, pour off ethanol, spin 1 min, and remove remaining ethanol without disturbing pellet. Dry pellet (air-dry or speed-vac).
- Resuspend pellet in 10 µL ddH2O and electroporate 3.3 µL of resuspended DNA according to a normal electroporation protocol. This electroporation protocol can yield up to 20 fold more colonies than chemical transformation of an equivalent TOPO-reaction. The addition of the 100 µL ddH2O followed by 10 mins incubation is not absolutely necessary, but it sufficiently dilutes the reaction and may help inactivate topoisomerase so that it is more easily electroporated.
We would suggest using our TOPO TA cloning kit for sequencing, which contains the pCR 4 TOPO vector, or our Zero Blunt TOPO PCR cloning kit for sequencing, which contains the pCR4Blunt-TOPO vector.
Due to the small size of your product, we recommend using the pCR 2.1 TOPO vector for your cloning. This size fragment would not be able to fully interrupt the ccdB gene in the pCR4-TOPO vector, and therefore, you may not get colonies as ccdB is lethal to E. coli.
Regular TOPO TA Cloning kits are efficient for cloning PCR products up to approximately 2-3 kb. With PCR products larger than 3 kb, the efficiency of cloning drops significantly. The TOPO XL PCR Cloning Kit has been optimized for TOPO cloning of long (3-10 kb) PCR products.
If using the regular TOPO kits, here are some tips to improve efficiency:
1. Use crystal violet instead of ethidium bromide (EtBr) to visualize the PCR for gel isolation to avoid DNA nicks
2. Increase incubation time of the TOPO reaction to 30 mins
3. Keep insert:vector molar ratio low, optimally 1:1
4. Dilute reaction to 20 µL, while maintaining same amount of vector and insert. Increase the volume of the salt solution to 3.7 µL to compensate for the increase in volume. Diluting the reaction reduces the competition for the vector ends.
Storage of the TOPO vector plus insert reaction for 1 week at 4 degrees C has shown no detectable decrease in the cloning efficiency of the TOPO reaction, as >95% of the colonies have insert. However, the total number of colonies was decreased by 10-fold. Storage of the TOPO reaction mix overnight at 4 degrees C showed little to no decrease in the number of colonies when compared to fresh TOPO reaction mix.
The composition of the 6X Stop solution is 0.3 M NaCl, 0.06 M MgCl2, and the composition of the 6X Salt solution is 1.2 M NaCl, 0.06 M MgCl2. Stop solution is only included in the TOPO XL Cloning kit whereas Salt solution is currently included in all of the other TOPO cloning kits. These solutions prevent free topoisomerase from re-binding and nicking the plasmid, which would reduce the number of colonies from a TOPO reaction.
When doing a TOPO cloning reaction, 2 µl of a PCR reaction containing up to 10% DMSO or 1.3 M betaine will not interfere with the TOPO reaction. Formamide and high levels of glycerol will inhibit the reaction. These reagents are usually added to the PCR reaction to enhance the yield of the PCR product, e.g., to reduce the effect of secondary structure or assist in amplification of GC-rich sequences. The effects of tricine or acetamide have not been tested on the TOPO cloning reaction.
PCR primers should not have 5'-phosphates when cloning into any TOPO vector, as the presence of 5'-phosphates inhibit the TOPO cloning reaction. Phosphorylated products can be TA-cloned but not TOPO-cloned. This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector. TOPO vectors have a 3' phosphate to which topoisomerase is covalently bound and a 5' phosphate. Non-TOPO linear vectors (TA and Blunt) have a 3' OH and a 5' phosphate. Phosphorylated products should be treated with phosphatase (CIAP) prior to TOPO-cloning. Treatment with CIAP may raise efficiency to 25%. PCR products generated with 5'-biotinylated primers (or any other 5'-label including 5'-Cy5) will not ligate into any of the TOPO vectors due to steric hindrance.
Gel purification is not required if the gel indicates that the PCR product is clean with no visible non-specific bands or primer dimers. It is recommended if the PCR product is >1.5 kb or if non-specific bands and primer dimers are visible on the gel. Smaller products clone much more efficiently into the vector than larger products; therefore, they should be eliminated from the sample prior to cloning. There is some reduction in A-overhangs if the PCR product is gel purified, which along with PCR product loss during the procedure may slightly reduce total number of colonies. However, the percentage of colonies with insert does not change; it is typically >90% recombinant clones.
For optimal TOPO cloning, we recommend using fresh PCR products.
TA cloning ligates the insert and vector using a T4 DNA ligase, while TOPO TA cloning uses the intrinsic properties of topoisomerase, which ligates the insert and vector during a 5 minute desktop reaction. TOPO TA cloning results in >95% recombinants, while TA cloning results in >80% recombinants.
We offer a custom service for TOPO cloning adaptation services. Our scientists can prepare your vector for either blunt TOPO cloning, TOPO TA cloning, or directional TOPO cloning of PCR products.
Yes, our pCR.1 TOPO TA (Cat. No. 450641), pCR4-TOPO TA (Cat. No. 450030), pCRBluntII-TOPO (Cat. No. 450245) are available separately.
No, we do not recommend this as these vectors contain the topoisomerase DNA protein complex conjugated to the end of the vector.
We suggest starting with a molar ratio of 1:1 (insert:vector), with a range of 0.5:1 to 2:1 (insert:vector). The ng quantities should be between 5-10 ng of a 2 kb PCR product in a TOPO cloning reaction.
Please consider the following before TOPO cloning:
- TOPO cloning cannot ligate DNA with a 5' phosphate group.
- TOPO cloning will decrease in efficiency inversely with the size of the insert (above 3 kb) unless using the TOPO XL cloning kit.
- TOPO vectors contain different antibiotic resistance markers which should be considered before purchase.
- TOPO TA vectors accept fragments containing a 3' A overhang while Zero Blunt vectors accept fragments that are blunt-ended.
TOPO and TOPO TA vectors (non-directional) have phenol red dye added. The color should be pink (or yellow) at room temperature. If it turns blue when PCR product is added, the PCR product buffer is too basic and the number of transformed colonies will drop. When the solution is yellow, it signifies an acidic pH. At a pH 2.0, TOPO vectors still maintain high cloning efficiency. Directional TOPO and Zero Blunt TOPO vectors have bromophenol blue dye added.
Subcloning DH5? cells are a compatible strain, but you will get lower efficiency (10e6 vs 10e9) and therefore risk getting fewer clones. Top10F' is also compatible, but if blue/white screening is performed, IPTG along with X-gal will be needed for detection due to the expression of the lacIq repressor present in cells containing an F' episome.
Ensure that you are using the correct antibiotic at the appropriate concentration. Additionally, make sure the antibiotic is not expired. If colonies exhibit unexpected morphologies, contamination could be a factor. Check your S.O.C. medium and LB growth medium.
Here are a few suggestions:
- Small fragments/linkers are cloning in to your vector instead of your insert; to correct this, gel-purify the insert before ligation
- Ensure that the correct concentrations of X-gal and/or IPTG (if vector contains the lacIq marker) are used
- If spreading X-gal and/or IPTG on your plate, allow sufficient time for the reagents to diffuse into the plate
- Incubate your plate for a longer period to ensure full color development
While there is no specific strain that works better with large plasmids, it is important to focus on transformation efficiency. For larger plasmids, chemically competent cells with highest efficiency are suggested, such as OmniMAX 2, TOP10, etc. We would recommend using an electrocompetent cell strain with plasmids larger than 20 kb for best efficiency.
With any strain, the first thing to try would be to lower the growth temperature of the culture to 30 degrees C or even lower (room temperature). Slower growth will generally allow E. coli to tolerate difficult sequences better. If reducing the growth temperature doesn't help, you may want to consider using a competent cell strain such as Stbl2 or Stbl4 cells, which have been shown to accommodate this type of sequence better than other strains in the same conditions.
We recommend trying the following:
- Carry out the puc19 transformation control; this gives you information about the performance of the cells.
- Check plates for expiration and correct media used (LB/agar).
- Confirm that the correct antibiotic and concentration was used.
The F' episome in TOP10F' has a lacIq marker, which over-expresses the lac repressor. IPTG must be added to LB plates along with X-gal to see beta-galactosidase expression and blue color in this strain. TOP10, on the other hand, does not require IPTG for blue/white screening.
There are a few conditions that can lead to this: SOC medium or other media used when plating was contaminated, DNA was contaminated with amp-resistant microbes, you used old plates with degraded amp, or the competent cells themselves were contaminated.
If you are using a mcr/mrr(+) competent cell strain, cellular enzymes may be recognizing eukaryotic methylation patterns on the yeast genomic DNA and deleting or rearranging it. Try a mcr/mr(-) strain such as Top10, DH10B, or OmniMAX 2.
XbaI cutting site is a Dam-methylation sensitive restriction site. TOP10 is a dam(+) strain, which means it expresses the methylating enzyme, Dam. You can try re-transforming into a dam(-) strain, such as INV110. Other dam- (and dcm-) sensitive restriction sites include the following:
- Dam: Bcl I, Cla I, Hph I, Mbo I, Mbo II, Taq I, Xba I, BspH I, Nde II, Nru I
- Dcm: Ava II, EcoO 109 I, EcoR II, Sau96 I, ScrF, Stu I, Aat I, Apa I, Bal I, Kpn I, ISfi I
1. Use pUC or pUC-based vectors that contain the portion of the lacZ gene that allows for ? complementation.
2. Select an E. coli strain that carries the lacZdeltaM15 marker.
3. Plate transformations on plates containing X-gal. Spread 50 µg of 2% X-gal or 100 microliters of 2% bluo-gal (both can be dissolved in DMF or 50:50 mixture of DMSO:water) on the surface of a 100 mm plate and let dry. Alternatively, add directly to the cooled medium (~50 degrees C) before pouring the plates at a final concentration of 50 µg/mL for X-gal and 300 µg/mL for bluo-gal. Plates are stable for 4 weeks at 4 degrees C.
4. If the strain used carries the lacIq marker, add IPTG to induce the lac promoter. Spread 30 µl of 100 mM IPTG (in water) on 100 mm plates. Alternatively, add the IPTG directly to cooled medium (~50 degrees C) before pouring the plates to a final concentration of 1 mM. Plates are stable for 4 weeks at 4 degrees C.
5. Do not plate E. coli on medium containing glucose if using X-gal or bluo-gal for blue-white screening. Glucose competes as a substrate and prevents cells from turning blue.
For long-term storage, preparation of glycerol stocks stored at -70 degrees C is recommended. Follow the protocol below:
1. Pick one colony into 5 mL LB broth or S.O.C. medium. Grow overnight at 37 degrees C.
2. Prepare glycerol solution: 6 mL of S.O.B. medium and 4 mL of glycerol.
3. Take one volume of cells and add one volume of glycerol solution and mix.
4. Freeze in ethanol/dry ice. Store at -70 degrees C.
Yes, this is possible. We recommend using saturating amounts of DNA (10 ng of each plasmid). Make sure that the origin of replication is different in each plasmid so that they can both be maintained in the cell. If the ori is the same, the plasmids will compete for replication and the one with even a slight disadvantage will be lost. Alternatively, cells with a resident plasmid can be electroporated with a second plasmid without “electrocuring” taking place.
In plates, we recommend 50 µg/mL X-gal and 1 mM IPTG (0.24 mg/mL). When spreading directly onto agar plates, we recommend 40-50 µl of 40 mg/mL X-gal (2% stock) in dimethylformamide and 30-40 µl of 100 mM IPTG on top of the agar. Let the X-gal and IPTG diffuse into the agar for approximately 1 hour. Do not plate on media containing glucose, as it competes with X-gal or bluo-gal and prevents cells from turning blue.
Competent cell efficiency is measured by transformation efficiency. Transformation efficiency is equal to the number of transformants, or colony forming units, per microgram of plasmid DNA (cfu/microgram).
Some suggestions that will help you to obtain the highest transformation efficiency are:
- Thaw competent cells on ice instead of room temperature; do not vortex cells.
- Add DNA to competent cells once thawed.
- Ensure that the incubation times are followed as outlined in the competent cell protocol for the strain you are working with; changes in the length of time can decrease efficiency.
- Remove salts and other contaminants from your DNA sample; DNA can be purified before transformation using a spin column, or phenol/chloroform extraction and ethanol precipitation can be employed.
DH5? cells are commonly used for routine cloning, but are mcr/mrr+, and therefore not recommended for genomic cloning. The TOP10 competent cells, on the other hand, contain mutated mcr/mrr, and therefore are a good choice for routine cloning and can be used for cloning of methylated DNA, such as eukaryotic genomic DNA. Our Mach1 strain is the fastest growing cloning strain that is T1 phage resistant.
These small colonies are most likely caused by degradation of the Ampicillin. The colonies are just untransformed cells that grow on LB with degraded Amp. In order to circumvent this scenario, you can try to:
1. Plate cells at a lower density
2. Use fresh LB-Amp plates or replace Ampicillin with carbenicillin.
3. The plates should not be incubated for more than 20 hours at 37 degrees C. Beta-lactamase, the enzyme produced from the Ampicillin-resistance gene, is secreted from the Amp-resistant transformants and inactivates the antibiotic in the area surrounding the transformant colony. This inactivation of the selection agent allows satellite colonies (which are not truly Amp-resistant) to grow. This is also true if carbenicillin is being used.
One possible explanation could be toxicity associated with the insert. This toxicity does not affect slow growing cells on solid medium but is much stronger in faster growth conditions like liquid medium.
Suggestions:
1. Use TOP10F' or any other strain with the LacIq repressor
2. Try using any other strain appropriate for cloning.
3. Lower growth temperature to 27 - 30 degrees C and grow the culture longer
4. Another possibility to explain lack of growth is possible phage contamination. In this situation we recommend using an E. coli strain that is T1 phage resistant like DH5alpha-T1R.
This may be caused by the instability of the insert DNA in TOP10 E. Coli. In this case, E.coli strains such as Stbl2, Stbl3, or Stbl4 have been shown to support the propagation of DNA with multiple repeats, retroviral sequences, and DNA with high GC content better than other strains.
Some possible causes and remedies are:
- Ligase function is poor. Check the age of the ligase and function of the buffer.
- Competent cells are not transforming. Test the efficiency of the cells with a control supercoiled vector, such as puc19.
- Both molecules were de-phosphorylated.
- Inhibition of ligation by restriction enzymes and residual buffer. Try transformation of uncut vector, clean up restriction with phenol, or carry out PCR cleanup/gel extraction before ligation.
- Incorrect antibiotic selection used. Check the plasmid and plates and make sure concentration of antibiotic used is correct.
If nothing above applies, low to no colonies may be due to instability of the insert DNA in your competent cells. In this case, E. coli strains such as Stbl2, Stbl3, or Stbl4 have been shown to support the propagation of DNA with multiple repeats, retroviral sequences, and DNA with high GC content better than other strains.
If working with a vector that contains the lac promoter and the LacZ ? fragment (for ? complementation), blue/white screening can be used as a tool to select for presence of the insert. X-gal is added to the plate as a substrate for the LacZ enzyme and must always be present for blue/white screening. The minimum insert size needed to completely disrupt the lacZ gene is >400 bp. If the LacIq repressor is present (either provided by the host cells, for example TOP10F', or expressed from the plasmid), it will repress expression from the lac promoter thus preventing blue/white screening. Hence, in the presence of the LacIq repressor, IPTG must be provided to inhibit the LacIq. Inhibition of LacIq permits expression from the lac promoter for blue/white screening.
TOPO vectors containing the LacZ-ccdB cassette allow direct selection of recombinants via disruption of the lethal E. coli gene, ccdB. Ligation of a PCR product disrupts expression of the LacZ-ccdB gene fusion permitting growth of only positive recombinants upon transformation. Cells that contain non-recombinant vector are killed upon plating. Therefore, blue/white screening is not required. When doing blue/white color screening of clones in TOPO vectors containing the LacZ-ccdB cassette, colonies showing different shades of blue may be observed. It is our experience that those colonies that are light blue as well as those that are white generally contain inserts. The light blue is most likely due to some transcription initiation in the presence of the insert for the production of the lacZ alpha without enough ccdB expressed to kill the cells and is insert dependent. To completely interrupt the lacZ gene, inserts must be >400 bp; therefore an insert of 300 bp can produce a light blue colony. A white colony that does not contain an insert is generally due to a spontaneous mutation in the ccdB gene.
A minimum insertion of 150 bp is needed in order to ensure disruption of the ccdB gene and prevent cell death. (Reference: Bernard et al., 1994. Positive-selection vectors using the F plasmid ccdB killer gene. Gene 148: 71-74.)
Strains that contain an F plasmid, such as TOP10F', are not recommended for transformation and selection of recombinant clones in any TOPO vector containing the ccdB gene. The F plasmid encodes the CcdA protein, which acts as an inhibitor of the CcdB gyrase-toxin protein. The ccdB gene is also found in the ccd (control of cell death) locus on the F plasmid. This locus contains two genes, ccdA and ccdB, which encode proteins of 72 and 101 amino acids respectively. The ccd locus participates in stable maintenance of F plasmid by post-segregational killing of cells that do not contain the F plasmid. The CcdB protein is a potent cell-killing protein when the CcdA protein does not inhibit its action.
If working with a vector that contains the lac promoter and the LacZ alpha fragment (for ? complementation), blue/white screening can be used as a tool to select for presence of the insert. X-gal is added to the plate as a substrate for the LacZ enzyme and must always be present for blue/white screening. The minimum insert size needed to completely disrupt the lacZ gene is >400 bp. If the LacIq repressor is present (either provided by the host cells, for example TOP10F', or expressed from the plasmid) it will repress expression from the lac promoter, thus preventing blue/white screening. Hence in the presence of the LacIq repressor, IPTG must be provided to inhibit the LacIq. Inhibition of LacIq permits expression from the lac promoter for blue/white screening. X-gal (also known as 5-bromo-4-chloro-3-indolyl β-D-glucopyranoside) is soluble in DMSO or DMF, and can be stored in solution in the freezer for up to 6 months. Protect the solution from light. Final concentration of X-gal and IPTG in agar plates: Prior to pouring plates, add X-gal to 20 mg/mL and IPTG to 0.1 mM to the medium. When adding directly on the surface of the plate, add 40 µl X-gal (20 mg/mL stock) and 4 µl IPTG (200 mg/mL stock).
No, these vectors do not contain a functional promoter to express your gene of interest. These vectors are typically for subcloning or sequencing.
TA Cloning:
- This cloning method was designed for use with pure Taq polymerases (native, recombinant, hot start); however, High Fidelity or Taq blends generally work well with TA cloning. A 10:1 or 15:1 ratio of Taq to proofreader polymerase will still generate enough 3' A overhangs for TA cloning.
- Recommended polymerases include Platinum Taq, Accuprime Taq, Platinum or Accuprime Taq High Fidelity, AmpliTaq, AmpliTaq Gold, or AmpliTaq Gold 360.
Blunt cloning:
- Use a proofreading enzyme such as Platinum SuperFi DNA Polymerase.
Directional TOPO cloning:
- Platinum SuperFi DNA Polymerase works well.
For the Directional TOPO Cloning Vectors, a PCR product must be generated by a proofreading enzyme to create a blunt product. Pfx50 or Accuprime Pfx and Accuprime Pfx Supermix from Thermo Fisher Scientific are recommended for use.
When cloning a Pfx-amplified PCR product, the insert to vector ratio is an important consideration. The PCR product generally needs to be diluted since Pfx generates a high concentration of product and using too much insert DNA can hamper the TOPO reaction. A 1:1 molar ratio of vector to insert (or about 2-10ng of insert) is recommended.
If you wish to use a polymerase mixture containing Taq polymerase and a proofreading polymerase, Taq must be used in excess with a 10:1 ratio of Taq to the proofreading enzyme to ensure the presence of 3´ A-overhangs on the PCR product. If you use polymerase mixtures that do not have enough Taq polymerase or a proofreading polymerase only, you can add 3' A-overhangs following PCR. See the vector product manuals for details.
Some examples of Taq blends that are compatible with TOPO TA Cloning are Platinum Taq DNA Polymerase High Fidelity and AccuPrime Taq DNA Polymerase High Fidelity.
Taq polymerase has a non-template-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3´ ends of PCR products. The linearized vector supplied in our TA Cloning kits have single, overhanging 3´ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.
Assuming that the primer is at a 50 nM final concentration and 50 mM final salt concentration, the melting temperatures are: M13 Forward (-20) Primer = 52.7 and the M13 Reverse Primer = 45.3. For use in the control PCR reaction we recommend using an annealing temperature of 56C.
Our vectors have not been completely sequenced. Your sequence data may differ when compared to what is provided. Known mutations that do not affect the function of the vector are annotated in public databases.
No, our vectors are not routinely sequenced. Quality control and release criteria utilize other methods.
Sequences provided for our vectors have been compiled from information in sequence databases, published sequences, and other sources.
We recommend using the pcDNA 3.4 TOPO TA Cloning Kit (Cat. No. A14697).
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
pcDNA 3.3-TOPO TA Cloning Kit (Cat. No. K830001) has been discontinued. As an alternative, you may use pcDNA 3.4 TOPO TA Cloning Kit (Cat. No. A14697).
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We recommend using the pcDNA3.4 TOPO TA vector (Cat. No. A14697) for optimal expression. This vector contains the native, full-length cytomegalovirus (CMV) promoter and a woodchuck posttranscriptional regulatory element (WPRE) downstream of the cloning site. Other CMV-based vectors, however, have been shown to express similarly to the pcDNA3.4 TOPO TA vector, thus any CMV-driven vector is a good choice for the ExpiCHO system.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.