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View additional product information for GeneArt™ pYES1L Vector with Sapphire™ Technology - FAQs (A13287)
30 product FAQs found
我们建议将克隆重新接种到新的平板上并再次进行菌落PCR。不要使用试剂盒提供的微珠破坏酵母细胞，那些微珠是用于进行E. coli 转化的。另外，在50 uL PCR反应中使用不超过0.5微升稀释后的酵母裂解物。
确保您的酵母转化在30摄氏度下孵育3天，以获得合适大小的克隆。
请参考以下建议：
•完全按照实验步骤要求进行转化。
•不要冻融或漩涡混匀MaV203酵母感受态细胞。
•使用CSM-Trp琼脂平板进行转化。
•要获得最佳结果，请使用新开瓶的DMSO。您也可以使用保存在-20摄氏度下的DMSO。
可以，您可以根据下列建议用GeneArt High-Order载体转换元件（货号A13291）将您的E. coli载体改造为酵母兼容的克隆载体用于 GeneArt High-Order遗传组装系统：
•使用DNA Oligo Designer在线工具，确认您的载体和GeneArt High-Order载体转换元件没有内部同源序列以避免在GeneArt High-Order遗传重组系统内使用您的改造载体时出现可能的重排。
•如果最终质粒要被转移到E. coli中的话，对于1个大于15 kb的最终质粒，请使用一个带有单拷贝或低拷贝复制起始点的载体。通常，低拷贝质粒的效能显著大于高拷贝质粒。
•避免在自己的载体上使用氯霉素抗性，因为转换元件是氯霉素抗性。
•在连接后（建议使用1:10的载体：插入片段比），使用连接混合物转化E. coli细胞并涂板于双抗性LB平板上（氯霉素加用户载体上的抗生素标记）。要线性化酵母兼容的克隆载体用于多片段组装，需要进行一次双酶切以避免单酶切后产生的回文序列造成的背景。
用于插入编辑的stitching寡核苷酸除了插入碱基之外必须含有和每个相邻片段重叠的30bp核酸序列（最大长度80 mer，包括20 mer的插入碱基）。请参见用户手册内的图表。注意：这适用于2片段组装和插入仅适用于内部连接。对于其他的无缝连接，请使用40 bp重叠序列（即一个80 mer的寡核苷酸）。
用于删除编辑的stitching寡核苷酸必须含有和每个相邻片段重叠的40bp核酸序列，重叠区域可以离开片段连接处6个核苷酸处开始匹配，从而在每个片段末端有6 bp的序列可以在转化介导的重组中被删除。请参见用户手册内的图表。注意：这适用于2片段组装和删除仅适用于内部连接。对于其他的无缝连接，请使用40 bp重叠序列（即一个80 mer的寡核苷酸）。
推荐您使用我们的Platinum PCR Supermix High Fidelity酶（货号12532-016）扩增最大5 kb的DNA片段用于通常的组装应用。如果您需要更高的PCR保真性，我们推荐您使用带有扩增和校对功能的酶。
大片段（>5kb）更容易在凝胶回收过程中受到损伤。因此，当使用PCR获得片段时，我们建议您在一次反应内组装多个小于5 kb的片段，而不是一个单独的大片段。另外，尽量减少DNA暴露于UV照射的时间和/或溴乙锭（EtBr）的使用量。如果您要将插入片段连入线性化的克隆载体，所有提供同源性的核酸序列必须位于引物的5'末端。如果您要连接相邻的两个片段，您可以将30至50 bp的同原序列分别置于不同片段（例如对于30 bp同源区的设计，将15 bp置于片段1的反向引物，15 bp置于片段2的正向引物，或者对于50 bp同源区的设计，将25 bp置于片段1的反向引物，25 bp置于片段2的正向引物）。请注意，您可以在相邻片段之间任意分配同源序列（例如对于30 bp的同源序列，可以使用上例中的15+15或20或20+10，25+5等等）。
pYES1L载体，一个9.4 kb线性化的YAC-BAC穿梭质粒，是在MaV203细胞中进行DNA片段组装的对照载体并且可用来将组装后的重组DNA分子转移到E. coli中。如果需要，您也可以将它用作克隆载体。酵母的复制起始位点是Chromosome II的中心粒（单拷贝，高效能YAC）。E. coli的复制起始点是F’ ori （单拷贝，高效能BAC）。壮观霉素可以用来进行E. coli筛选。试剂盒内包含可用于4次反应（1管，8 µL）的pYES1L载体。这对于对照反应或克隆您的插入片段，或者将两者结合都是足够的。pYES1L载体也可以单独出售（货号A13287，10次反应）。
不会，您可以使用产生平末端或末端加A的酶。克隆效率不受影响。
克隆效率根据带有末端同源性的片段数目的不同而有很大的不同。请参见下列对于将片段组装进pYES1L载体时克隆效率的大致估计：
5个DNA片段每个10 kb， > 90%
10个DNA片段每个5 kb， > 90%
10个DNA片段每个10 kb， > 50%
对于使用“Stitching”DNA寡核苷酸的方法将已经存在的无末端同源性的片段组装进入pYES1L载体，通常的克隆效率如下：
1个10 kb片段， > 90%
2个片段每个10 kb， > 75%
在线工具不能自动进行片段编辑，但是可以用它来手动进行片段编辑。用户手册中有一个章节是关于片段编辑的，并且给出了如何使用在线工具进行片段编辑的示例。
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。
可以，试剂盒可以对最多5个片段和3个寡核苷酸补丁（Stitches）进行组装。
我们开发试剂盒时使用的是脱盐的寡核苷酸，但是更高的纯度将稍微提高克隆效率，特别是在进行片段编辑时。
对大于60 kb的片段我们的建议是至少设计与相邻片段有50个核苷酸的重叠区域。对小于60 kb的片段，建议使用30个核苷酸的重叠区域。
We would recommend trying to re-streak the colony on a fresh plate and repeat colony PCR. Do not break open the yeast cells with the beads supplied with the kit; the beads are for transformation into E. coli. Additionally, use less than 0.5 µL of diluted yeast lysate in a 50 µL PCR reaction.
Ensure that yeast transformations are incubated at 30 degrees C for 3 days for proper colony formation.
Please review the following suggestions:
– Perform transformation exactly as described in protocol.
– Do not freeze-thaw or vortex MaV203 yeast competent cells.
– Use CSM-Trp agar plates for the transformation.
– For best results, use fresh DMSO from an unopened bottle. You may use DMSO stored at -20 degrees C.
Yes, you should be able to adapt your E. coli vector into a yeast-compatible cloning vector using the GeneArt High-Order Vector Conversion Cassette (Cat. No. A13291) for use with the GeneArt High-Order Genetic Assembly System with the following provisions:
– Start by using the DNA Oligo Designer web tool, and verify that your vector and the GeneArt High-Order Vector Conversion Cassette do not share internal homology to prevent potential re-arrangements when using your adapted vector with the GeneArt High-Order Genetic Assembly System.
– Use a vector with a single- or low-copy-number origin for a final construct of >15 kb, if the final plasmid construct will be transferred into E. coli. Usually, low-copy-number E. coli vectors have significantly higher capacity than high-copy number vectors.
– Avoid chloramphenicol selection markers on the custom vector since this is the marker on the cassette.
– After ligation (1:10 vector: insert ratio recommended), transform competent E. coli cells with the ligation mixture and plate on double selection LB plates (chloramphenicol plus the antibiotic marker on your custom vector backbone).To linearize your yeast-adapted cloning vector for multi-fragment assembly, a double-digestion is required to avoid background caused by residual palindromic end sequences resulting from a single enzyme digestion.
Stitching oligonucleotides used for insertion editing must have a 30-nucleotide overlap with each adjacent fragment in addition to the insertion bases (for a total length of up to 80-mer, including up to 20 insertion bases). See manual for diagram. Note: This applies for a 2-fragment assembly and the insertion applies only to the internal junction. Use a 40-bp overlap (i.e., an 80-mer oligonucleotide) for the remaining seamless junctions.
Stitching oligonucleotides used for deletion editing must have a 40-nucleotide overlap with each adjacent fragment, annealing up to 6 nucleotides from the junction into each fragment, thus leaving up to 6 bp at the end of each fragment to be deleted during transformation-associated recombination. See manual for diagram. Note: This applies for a 2-fragment assembly and the deletion applies only to the internal junction. Use a 40-bp overlap (i.e., an 80-mer oligonucleotide) for the remaining seamless junctions.
We recommend using our Platinum PCR Supermix High Fidelity enzyme (Cat. No. 12532-016) for amplifying DNA fragments up to 5 kb for general assembly applications. If you require higher PCR fidelity, we would recommend one that has processing and proof-reading capabilities.
Large fragments (>5 kb) are more susceptible to damage in a gel extraction procedure. Therefore, when using DNA fragments generated by PCR, we recommend that you assemble multiple fragments of less than 5 kb in one reaction rather than a single large fragment. Additionally, try to minimize UV exposure of the DNA and/or limit EtBr amount used. If you are attaching the insert to the linearized cloning vector, all nucleotides providing the requisite homology must be on the 5' end of the primer. If you are connecting two adjacent inserts, you may split the 30- to 50-bp homology between the fragments (e.g., 15 bp on the reverse primer of fragment 1 and 15 bp on the forward primer of fragment 2 for a 30-bp homology, or 25 bp on the reverse primer of fragment 1 and 25 bp on the forward primer of fragment 2 for a 50-bp homology). Please note, you can split the homology between adjacent fragments in any combination (e.g., 15 + 15 as in the example above or 20 + 10, 25 + 5 etc. for a 30-bp homology).
The pYES1L vector, a 9.4 kb linearized YAC-BAC shuttle plasmid, is a control vector for the assembly of DNA fragments in MaV203 cells and for the transfer of the assembled recombinant DNA molecule into E. coli. You can also use the pYES1L vector as a cloning vector, if desired. The origin of replication for yeast is chromosome II's centromere (single copy, high capacity YAC). The origin of replication for E. coli is F' ori (single copy, high capacity BAC). Spectinomycin is used for selection in E. coli. Four reactions (1 tube with 8µL) of the pYES1L vector are included with the kit. This is enough for control reactions, for cloning your inserts, or a combination of both. The pYES1L vector is also available separately (Cat. No. A13287, 10 reactions).
No, you can use enzymes that leave blunt ends or A-overhangs. Cloning efficiency will not be affected.
Cloning efficiencies can vary greatly based on the number of fragments with end-homology. Below are the approximate cloning efficiencies of assembled fragments into pYES1L:
>90% for 5 DNA fragments of 10 kb each
>90% for 10 DNA fragments of 5 kb each
>50% for 10 DNA fragments of 10 kb each
For pre-existing fragments without end-homology assembled into pYES1L using ‘stitching' DNA oligonucleotides, common cloning efficiencies are:
>90% for 1 fragment of 10 kb
>75% for 2 DNA fragments of 10 kb each
The web tool is not set up to do fragment editing automatically, but it can be used to do it manually. The manual has a section on fragment editing that gives examples on how to use the tool for this purpose.
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.
Yes, the kit can assemble up to 5 fragments and 3 oligo stitches.
We have developed the kit using desalted oligonucleotides, but higher purities may result in slightly better cloning efficiencies, especially during fragment editing.
We recommend having a total overlap of 50 nucleotides with adjacent fragments for constructs larger than 60 kb. For constructs smaller than 60 kb, a total overlap of 30 nucleotides is recommended.