Question 1

什么是校正酶（CorrectASE）？它的功能是什么？

Accepted Answer

CorrectASE是一种酶，它可以消除由寡核苷酸合成错误导致的错配，导致合成基因或片段的错配减少3 - 10倍。它作为一个独立的产品出售（货号A14973），或作为GeneArt基因合成试剂盒(货号A14971）的一部分。

Question 2

我对Do It Yourself 基因合成试剂盒所用的引物浓度有点困惑，请对这个问题做一些说明。

Accepted Answer

根据实验方案，寡核苷酸储存液应该用1X TE缓冲液配制，其终浓度为100 μM。下一行建议将浓度为10 μM的引物各取5μl加到一起。根据研发同事的反馈，操作手册这样写是因为他们通常将冻干的寡核苷酸配制成浓度为100 μM的储液（由于EP管体积的原因）。但是你做实验时可能会需要用到浓度为0.15 μM的引物pool。这时，你可以将储液稀释到10 μM或者将引物pool按1:10稀释。

Question 3

使用GeneArt Do-It-Youself基因合成试剂盒时，我没有得到PCR产物，我应该怎么办？

Accepted Answer

使用CorrectASE酶过度酶切可能会导致DNA模板降解。

确保反应不超过60分钟。同时，确保在PCR步骤前反应物保持冰浴，否则反应物将有可能被CorrectASE酶过度酶切。

Question 4

我觉得我的序列不准确，应如何处理？

Accepted Answer

请发送电子邮件到geneartsupport@lifetech.com对有错误的地方进行说明。请同时提供项目编号及载体编号（ID），以便我们将问题反馈给质检部门做进一步调查。

Question 5

我不能使用Xba1对GeneArt合成的我的感兴趣的基因进行酶切。这是什么原因？

Accepted Answer

我们在生产过程中会采用不同的菌种，例如Top10或DH5α。一般情况下，我们用dam+菌种进行培养。因此，你可能发现酶切受到抑制，因为Xba1对dam甲基化敏感。

Question 6

What does CorrectASE enzyme do?

Accepted Answer

The CorrectASE enzyme has the ability to remove mismatches caused by oligonucleotide synthesis errors and is typically used in a do-it-yourself gene synthesis workflow to aid in decreasing mutations in your synthetic genes/fragements.

Question 7

I'm confused with the primer concentration to use for the Do It Yourself Gene Synthesis Kit. Can you please clarify?

Accepted Answer

The protocol states that oligonucleotide stocks should be prepared at a final concentration of 100 µM in 1X TE buffer. The next line indicates the addition of 5 µL of each 10 µM primer together. According to R&D, the manual was written this way because our R&D typically brings up the lyophilized oligos to a 100 µM stock concentration (due to the volume of the tube). You do, however, want to use a 0.15 µM pool. Therefore, you can either dilute the stock to 10 µM or dilute the primer pool 1:1

Question 8

I am not getting a PCR product using your GeneArt Do-It-Yourself Gene Synthesis Kit. What should I do?

Accepted Answer

Overdigestion with CorrectASE enzyme can lead to degradation of the DNA template.

Ensure that the reaction does not go longer than 60 mins. Also, ensure that the reaction is kept on ice until the PCR step or else the reaction will be prone to overdigestion by the CorrectASE enzyme.

Question 9

I don't think my sequence is accurate. What should I do?

Accepted Answer

Please send an email to geneartsupport@lifetech.com explaining what is wrong. We will also need the project ID and construct ID, which we will forward to our QC department for further investigation.

Question 10

After GeneArt Gene Synthesis, I cannot cut out my gene of interest using Xba1. Why is this?

Accepted Answer

We have a variety of strains that are used in production, such as Top10 or DH5α. Routinely, we grow them in dam+ strains. Therefore, you may be seeing inhibition because Xba1 is sensitive to dam methylation

CorrectASE™ Enzyme - FAQs