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View additional product information for GeneArt™ Site-Directed Mutagenesis System - FAQs (A13282)
26 product FAQs found
GeneArt定点突变试剂盒内的DNA甲基化酶不作为单独产品出售。
当在环形模板上进行第一个PCR循环时，聚合酶将生成两个能够退火并在下一个循环能进行引物延伸的线性产物（参见下图）。在两个末端引入了突变的PCR产物将在后续循环中累积。在PCR之后，PCR产物经过重组反应进行环化，从而进一步提高了突变效率。
引物长度在30-45 nt左右，突变位置应位于引物中央。根据突变的大小，突变两侧应至少有15-20 nt的侧翼序列，引物的长度不超过45 nt。我们建议使用我们的在线GeneArt引物设计工具。
浓度是32 mM。
•GeneArt试剂盒合并了甲基化和DNA扩增步骤，实验方案节省了1小时
•GeneArt 无缝克隆和组装试剂盒内的酶混合物是实验方案的一部分，可以大大提高了克隆的效率而无需进行很多轮PCR扩增（这是另一个节省时间的步骤）
•新试剂盒还包含一个应用于PCR步骤的增强子，可以提升突变效率，适用的质粒大小范围很广。
两种试剂盒几乎相同，但是PLUS试剂盒包含一个改进的2X GeneArt酶混合物，并提供了针对多点突变优化的protocol （最多3个位点，所用质粒最大14 kb）。PLUS试剂盒还可以用于在小于14 kb的质粒内进行最多25个核苷酸的单点突变，而原来的试剂盒只能用于最多12个核苷酸的替换/删除/插入。有一个网络工具可以用于引物设计，该工具也适用于原来的试剂盒。
可以。将“single variant”作为新过程添加，然后选择“upstream service”（“gene synthesis”，“already at Geneart”，或者“I will provide this master gene”）。如果您提供的是您自己的基因，那么您将需要填写载体+插入片段的全部序列。如果它只是一个质粒，您将需要将突变区域填写为“insert”，而将质粒剩余部分作为“vector”。“插入片段”最长可以是4 kb。
是的，我们提供三种定点突变试剂盒：GeneArt定点突变系统和GeneArt定点突变PLUS试剂盒、Phusion定点突变试剂盒以及GeneArt定制服务。< br / > < br / > 这些试剂盒几乎是相同的，但是PLUS试剂盒包含了改良的2倍GeneArt酶混合物，以及针对多位点突变优化的方案(质粒中最多有3个位点，最长可达14kb)。PLUS试剂盒还可用于多达25个核苷酸的单位点突变，而原始试剂盒可在长达14kb的质粒中进行的单碱基替换/缺失/插入最多是12个核苷酸。另外还引入了一个web工具用于引物设计，但它也可以用于原来的试剂盒。Phusion定点突变试剂盒是一种多功能、高效的工具，可以在任何类型的质粒DNA中引入点突变、插入或缺失。使用这个试剂盒，整个质粒被磷酸化引物扩增，引物引入所需的变化。包含目的突变的线性PCR扩增产物，在T4 DNA连接酶作用下进行连接反应5分钟被环化。得到的质粒可以转化任何大肠杆菌感受态细胞中。
下面是引起此问题的一些典型原因及解决方法：
•DNA甲基化酶失活或SAM失活：利用甲基化对照反应测试DNA甲基化酶及25X SAM的活性。
•使用了太多的DNA：每50 μL甲基化反应体系中DNA的量不要超过50 ng。
•过度扩增：将小质粒的PCR循环数降到12，或将中等长度的质粒的循环数降到15。
下面列举了引起这个问题的典型原因及解决方法：
•DNA量太少：每50 μL反应最多可以加入50 ng DNA。
•DNA质量差：重新纯化质粒。
•使用了不合适的DNA聚合酶：我们推荐使用1个单位的AccuPrime Pfx DNA聚合酶进行扩增。
•PCR条件不合适：优化退火温度及延伸时间。
•引物设计不合适：使用GeneArt引物与载体设计工具降低可能的二级结构或增加引物长度。
请参考以下建议：
•您使用的是什么聚合酶？对于两种突变试剂盒，我们都推荐使用AccuPrime Pfx DNA聚合酶——使用1个单位的聚合酶进行扩增。
•尝试优化退火温度及延伸时间。按经验法则，采用的退火温度一般是比引物的最低Tm值低5至10摄氏度，延伸时间按照每30秒延伸1kb来计算。您可以尝试不同的温度/时间优化您的反应。
•引物设计不合适会造成没有反应产物。我们推荐使用我们的工具降低可能的二级结构或增加引物长度。
•检查您使用的起始DNA样本质量。
•确保您在PCR反应中加入了足够量的DNA。
We do not offer the DNA methylase from GeneArt Site-Directed Mutagenesis kits as a separate product.
During the first cycle of PCR on the circular template, the polymerase will generate two linear products that are able to anneal and undergo primer extension in the next cycle. PCR products that incorporate the mutation at both ends will accumulate in subsequent cycles. After PCR, the recombination reaction results in circularization of the PCR product which enhances the mutagenesis efficiency.
The primer should be 30-45 nucleotides in length, with the mutation centrally located. The mutation should be flanked by at least 15-20 nucleotides on either side, depending on the size of the mutation, with the primer length no longer than 45 nucleotides. We recommend using our online GeneArt primer design tool.
The concentration is 32 mM.
The differences are as follows:
- The GeneArt kit combines the methylation and DNA amplification steps, saving 1 hour in the protocol.
- The enzyme mix from the GeneArt Seamless Cloning and Assembly Kit is part of the protocol, boosting the colony output without the need to do many PCR cycles (another time saving step).
- The new kit also contains an enhancer used during the PCR step that increases mutagenesis efficiency for a wide range of plasmid sizes.
The kits are almost the same, but the PLUS kit contains an improved 2X GeneArt enzyme mix with protocols optimized for multi-site mutagenesis (up to 3 sites in plasmids up to 14 kb). The PLUS kit can also be used for single-site mutations of up to 25 nucleotides, while the original kit can be used for single substitution/deletion/insertions up to 12 nucleotides in plasmids up to 14 kb. A web tool is available for primer design that can also be used for the original kit.
Yes. Add “single variant” as a new process, then choose an “upstream service” which is: gene synthesis, already at Geneart, or I will provide this master gene. If the customer is providing their own gene, they will need to fill in the field for the complete sequence of their vector + insert. If it is a plasmid only, they will put the mutagenized area as the “insert” and the rest of the plasmid as the “vector”. The “insert” can only be up to 4kb long.
Yes, 25X SAM is not stable and will lose activity within a few hours. We recommend creating a new dilution of SAM from the 200X SAM supplied in the kit each time you perform a mutagenesis procedure.
We recommend using AccuPrime Pfx DNA Polymerase with the GeneArt Site-Directed Mutagenesis kits.
The kits are almost the same, but the PLUS kit contains an improved 2X GeneArt enzyme mix with protocols optimized for multi-site mutagenesis (up to 3 sites). A web tool was also introduced for primer design, but this can also be used for the original kit.
Yes, we offer three kits for site-directed mutagenesis: GeneArt Site-Directed Mutagenesis System and GeneArt Site-Directed Mutagenesis PLUS kit, Phusion Site-Directed Mutagenesis Kit as well as a custom GeneArt service.
The kits are almost identical, but the PLUS kit contains an improved 2x GeneArt Enzyme mix with protocols optimized for multi-site mutagenesis (up to 3 sites in plasmids up to 14 kb). The PLUS kit can also be used for single-site mutations of up to 25 nucleotides, and the original kit can be used for single substitution/deletion/insertions up to 12 nucleotides in plasmids up to 14 kb. A web tool was also introduced for primer design, but this can also be used for the original kit. Phusion Site-Directed Mutagenesis Kit is a versatile and efficient tool for introducing point mutations, insertions, or deletions in any type of plasmid DNA. With this kit, the entire plasmid is amplified using phosphorylated primers that introduce the desired changes. The amplified, linear PCR product, containing the desired mutation, is circularized in a 5-minute ligation reaction with T4 DNA Ligase. The resulting plasmid can be then transformed into any competent E. coli cells.
The detailed mechanism is still not very clear, but involves the E. coli double strand break repair system. The key steps are:
(1) An exonuclease, like RecBCD, removes nucleotides from the ends to generate a single-stranded region.
(2) Strand invasion with homologous regions occurs.
(3) Repair enzymes including nucleases, ligase, and DNA polymerases repair gaps and/or nicks.
Please see the typical causes and solutions for this problem below:
-Inactive DNA methylase or inactive SAM: Test the activity of the DNA methylase and 25X SAM using the methylation control reaction.
-Too much DNA used: Use no more than 50 ng of DNA per 50 µL of methylation reaction.
-Over-amplification: Reduce PCR cycles to 12 for small plasmids or 15 for intermediate size plasmids.
Please see the typical causes and solutions for this problem below:
-Too little DNA: use up to 50 ng DNA per 50 µL reaction.
-Poor quality DNA: purify new plasmid.
-Incorrect DNA polymerase used: We recommend using 1 unit of AccuPrime Pfx DNA Polymerase for amplification.
-PCR conditions were incorrect: Optimize annealing temp and extension time.
-Poor primer design: Use the GeneArt Primer and Construct Design Tool to reduce potential secondary structures or increase primer length
Please review the following recommendations:
-What polymerase are you using? We recommend using AccuPrime Pfx DNA polymerase with both mutagenesis kits - use 1 unit of the polymerase for amplification.
-Try optimizing the annealing temperature and extension time. The rule of thumb is 5-10 degrees C below your primer's lowest melting temperature for the annealing temperature, and 30 seconds per 1kb for extension time. You can experiment with different temperatures/times to optimize your reaction.
-Poor primer design could result in no product. We recommend using our tool to reduce potential secondary structures or increase the primer length.
-Check the quality of your starting DNA sample.
-Ensure that you are adding enough DNA to the PCR reaction