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View additional product information for GeneArt™ Seamless Cloning and Assembly Kit - FAQs (A13288)
30 product FAQs found
对于单独的片段大小没有限制，只要重组后4个单独片段加上载体的总长度不超过13 kb。
使用凝胶电泳检查您的PCR产物。您可能得到了多个PCR产物，导致在您的载体中连入了错误的插入片段。对PCR产物进行凝胶纯化能帮助解决这一问题。
检查以确保克隆载体的线性化是完全的。另外，GeneArt酶混合物加入的顺序对实验很关键 - 请最后加入酶混合物。最后，检查反应的孵育时间是否是推荐的时间。
请参考下面的建议：
•检查PCR产物的纯度。
•确保片段末端之间存在所需的末端同源序列。
•DNA末端可能在制备过程中被破坏了（例如暴露于UV照射），请减少DNA暴露于UV/EtBr的时间。
•检查DNA插入片段和载体之间的比例（插入片段：载体摩尔比2:1）。
•确保正确操作GeneArt 酶混合物。该酶是温度敏感的。请在使用后立刻放回存储位置。不要将其放在室温或冰上过长时间。
我们不建议使用电转化感受态细胞。酶混合物不进行DNA末端的连接，所以电转化会破坏组装中形成的DNA碱基对。
很不幸，GeneArt 克隆和组装试剂盒里的10X酶混合物在-20摄氏度下不稳定，并会在此温度下迅速失去活性。酶混合物在-20摄氏度保存两个月后其产出的克隆数量会大大下降。
如果这些片段全部小于5 kb且分子的总长度小于13 kb，那么我们建议使用GeneArt Seamless克隆和组装（PLUS）试剂盒。如果您组装的片段没有末端同源性，或者由于太大而不能进行PCR扩增，或者组装后的分子大于13 kb，那么我们推荐使用GeneArt High-Order组装系统。总体来说，High-Order 系统可以组装更多片段，进行片段编辑，以及进行寡核苷酸拼接并且效率更高。但是，它的组装是在体内（酵母中）进行并且得到最终质粒需要更长的时间，因为酵母的生长周期比E. coli长。请使用这一表格(https://www.thermofisher.com/cn/zh/home/life-science/cloning/seamless-cloning-and-genetic-assembly.html)来选择最适合您应用的GeneArt Seamless克隆和组装试剂盒。
这不会是一个问题。比推荐的长度少几个碱基的重叠区域仍然会在反应中正常发挥作用。但是，获得最佳结果的重叠区域是15 bp。
我们测试过的最小片段是100 bp（>95%的克隆含有插入片段）。我们没有试过使用退火后的寡核苷酸作为插入片段。
我们不建议这么做，因为克隆效率/克隆产出会降低。这里有一些增加大片段组装成功率的诀窍：
•确保载体背景较低——酶切载体，胶回收，然后PCR扩增载体。如果不能进行PCR扩增，可以再进行一次纯化以避免凝胶纯化后对反应的抑制。
•试试将载体和片段之比从1:2改为1:1。
•转化时不要用超过6 µL的反应产物，并且不要用OmniMAX 2细胞，尽管它们的克隆效率较高。TOP10和MAX Efficiency DH5alpha感受态细胞转化效率最高。
•在加入SOC后孵育更长时间（2小时）。使用950 µL SOC，37摄氏度孵育2小时，然后离心沉淀细胞。去除大约800µL，将剩下的部分进行涂板。
•较长的重叠区域（例如80 nt）有利于大片段重组。如果片段的末端之间具有较长的重叠区域，那么孵育45分钟到1小时可能提高克隆效率。但是，较长的孵育时间对于小片段（300 bp）的重组则可能有负面影响——重组酶将过多回切片段的末端。
我们不建议过度孵育，因为酶混合物会回切，导致缺失突变。较短的孵育时间（例如20分钟）可能是可行的。对于4个片段和1个载体，我们试过15摄氏度，室温，以及30摄氏度，而最佳结果是在室温获得，克隆效率达77%。另外两个温度的克隆效率分别是31%和37%。我们不建议在冰上孵育，因为这可能会导致在连接处产生很多缺失突变。
我们推断短片段的效率可能会高一些。对于一个5 kb片段我们得到过100%的克隆效率，但是克隆数比2 kb片段少。例如，对于5 kb片段1 uL反应可获得大约400个克隆，而对于2 kb片段1 uL反应可获得大约1200-2000个克隆。另外，我们还观察到，在类似5 kb的大片段组装过程中，如果没有对该5 kb片段进行凝胶纯化并且有一个明显的较小PCR条带，那么较小的片段比5 kb片段更容易被克隆进载体。对于1 kb或2 kb的片段我们没有观察到类似现象。
理论上，GeneArt Seamless克隆系统可以用于文库构建，但是我们没有测试两种中任何一种应用。要进行文库构建需要生成带有克隆载体同源序列的接头。
我们建议GeneArt Seamless克隆至少要有15 bp的同源区域，对于较大的插入片段我们也尝试过40或80 bp。
GeneArt Seamless PLUS试剂盒是原来试剂盒的升级版。它被推荐用于4片段组装并且最大可组装40 kb的载体，而原来的试剂盒最大可组装的载体仅为13 kb。GeneArt酶混合物以带有缓冲液的2X混合物形式提供，可以存储于-20摄氏度而不是-80摄氏度。最后，试剂盒包括线性化的pYES7L载体和Stbl3/pRK2013感受态细胞，可以将载体横向转移到很多种受体菌株内。使用此处表格(https://www.thermofisher.com/cn/zh/home/life-science/cloning/seamless-cloning-and-genetic-assembly.html)以选择最适合于您的应用的GeneArt Seamless克隆和组装试剂盒。
There is no size limit on the individual fragments as long as the combined total length of the 4 individual fragments and vector does not exceed 13 kb.
Check your PCR product on a gel. You may be getting multiple products, causing incorrect inserts to clone in to your vector. Gel purification of the PCR products can help with this issue.
Check to ensure that the cloning vector is completely linearized. Additionally, the order in which the GeneArt Enzyme mix is added is crucial to the experiment – add it last. Lastly, check the incubation time of the reaction for the recommended time.
– Check the purity of the PCR products.
– Ensure that the required end-terminal homology between ends is present.
– DNA ends may have been damaged during preparation (exposure to UV); limit exposure time to UV/EtBr.
– Check ratio and amounts of DNA inserts and vector (2:1 insert:vector molar ratio).
– Ensure that the GeneArt Enzyme Mix is handled correctly.This enzyme is temperature sensitive. Return immediately to storage after use. Do not leave at room temperature or on ice for extended periods.
We do not recommend using electrocompetent cells. The enzyme mix does not perform ligation of the DNA ends, so electroporation will disrupt the DNA base pairs formed during assembly.
Unfortunately, the 10X Enzyme Mix in the GeneArt Seamless Cloning and Assembly Kit is unstable at -20 degrees C, and will quickly lose activity at this temperature. We have seen a large drop in colony output after storing the enzyme mix at -20 degrees C for 2 months.
If the fragments are all below 5 kb and the total size of the molecule is below 13 kb, we would recommend the GeneArt Seamless Cloning and Assembly (Plus) kit. If you are assembling elements that have no end-homology, are too large to be amplified by PCR, or are trying to create a molecule over 13 kb, we recommend the GeneArt High-Order Genetic Assembly System. Overall, the High-Order system can do more fragments, fragment editing, and oligonucleotide stitching and can do it at a higher efficiency. However, the assembly is in vivo (yeast) and takes longer to get the final construct due to longer growth periods for yeast compared to E. coli. Select the GeneArt Seamless Cloning and Assembly kit that is best for your application by visiting http://www.thermofisher.com/us/en/home/life-science/cloning/seamless-cloning-and-genetic-assembly.html.
This should not be a problem. An overlap that is a few bases shorter than recommended should still function in the reaction. However, for best results always use 15 bp.
The smallest insert size we have tested is 100 bp (>95% colonies contained insert). We haven't tried annealed oligos for this.
We do not recommend this as cloning efficiency/colony output can decrease. Here are some tips to increase the likelihood of a larger assembly working:
– Make sure vector background is low - RE cut the vector, gel purify, then PCR amplify the vector. If PCR not possible, you can do a second cleanup to avoid inhibition after gel purification.
– Try a ratio of 1:1 instead of 1:2.
– Do not transform more than 6 µL, and do not use OmniMAX 2 cells even though they have a higher efficiency. TOP10 and MAX Efficiency DH5alpha work best.
– Try a longer recovery time (2 hours) after addition of SOC. Use 950 µL SOC, incubate for 2 hours at 37 degrees C, and then spin down the cells. Remove ~800 µL and plate the rest on one plate.
– Longer overlaps (80 nt, for example) are better for large constructs. If the fragment ends have long overlaps, it may work better to try incubating for 45 min - 1 hour. However, small fragments (300 bp) may be negatively affected by this longer incubation - the enzyme will chew back the ends too much.
We don't recommend over-incubating since the enzyme mix may chew back too much, resulting in deletions. Shorter incubation times (e.g., 20 min) may be okay. For 4 fragments and 1 vector, we have tried 15 degrees C, RT, and 30 degrees C, and the best results were at RT with 77% cloning efficiency. The other temperatures gave us 31% and 37% efficiency. We do not recommend incubating on ice as you may get a lot of deletions at the junctions.
We suspect that there would be some degree of preference for shorter fragments. We have seen 100% cloning efficiency with a 5 kb fragment, but the colony output was lower when compared to a 2 kb fragment. For example, you get about 400 colonies per 1 µL reaction for 5 kb and about 1200-2000 colonies per 1 µL for 2 kb. Also, we have observed in assemblies of larger fragments like 5 kb that if the PCR reaction of the 5 kb fragment is not gel-purified and there is a significant PCR band at a smaller size, then the smaller fragment tends to go in more than the 5 kb. We have not observed anything like this in fragments of 1 kb or 2 kb.
In theory, the GeneArt Seamless Cloning system can be used for library construction but we have not tested either application. Adapters with the required homology to the cloning vector would have to be generated.
We recommend at least 15 bp of homology for your GeneArt Seamless cloning and we have also tried 40 or 80 bp for larger inserts.
The GeneArt Seamless PLUS kit is an improved version of the original kit. It is recommended for the assembly of up to 4 fragments and a vector totaling up to 40 kb compared to 13 kb for the original. The GeneArt enzyme mix is also provided as a 2X mix with buffer that can be stored at -20 degrees C instead of -80 degrees C. Finally, the kit comes with the linearized pYES7L vector and Stbl3/pRK2013 cells that allows for horizontal transfer of the construct into a variety of recipient strains. To select the GeneArt Seamless Cloning and Assembly kit that is best for your application, please visit https://www.thermofisher.com/us/en/home/life-science/cloning/seamless-cloning-and-genetic-assembly.html.