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PfxUltima with pENTR™/TEV/D-TOPO® Cloning Kit with One Shot® TOP10 Chemically Competent E. coli - FAQs

查看更多产品信息 PfxUltima with pENTR™/TEV/D-TOPO® Cloning Kit with One Shot® TOP10 Chemically Competent E. coli - FAQs (12355061)

6 个常见问题解答

Gateway兼容的TOPO载体同时提供一个对照模板和对照引物。可否提供对照模板的序列吗？

对照模板的序列是受专利保护的。

我的定向TOPO克隆反应效率很低，如何解决这一问题？

这里是一些提高定向TOPO克隆反应效率的建议：

•确保 5’引物带有–CACC而3’引物不含有类似于GTGG的序列。
•PCR产物：TOPO载体的摩尔比是成功的关键。我们建议使用1:1到2:1的摩尔比，从PCR产物：TOPO载体比值为1:1开始。当PCR产物：TOPO载体的摩尔比<0.1:1或>5:1时TOPO克隆效率将显著下降。这些结果通常是由于在TOPO克隆反应中使用了太少的PCR产物（例如PCR产物稀释过多）或者使用了过多的PCR产物造成的。如果对PCR产物的产量进行了定量，那么可能需要在进行TOPO克隆前对PCR产物浓度进行调整。对于pENTR TOPO载体而言，在TOPO克隆反应中使用1-5 ng的1 kb PCR产物或5-10 ng的2 kb PCR产物通常会得到较多数量的克隆。

我正在寻找带有Gateway兼容的att位点的克隆载体以便将来转移至表达载体。定向克隆偏好将是一个很大的优势。你们有什么建议吗？

我们建议使用我们的pENTR D-TOPO试剂盒能兼容Gateway系统，轻松实现TOPO克隆的同时具有定向性优势。

Your Gateway-adapted TOPO vectors are supplied with a control template and control primers. Can I obtain the sequence of the control template?

The sequence of the control template is proprietary.

I'm getting low cloning efficiency with my directional TOPO cloning reaction. What should I do?

Here are suggestions to try to increase your cloning efficiency with dTOPO cloning:

- Ensure that the 5' primer has CACC and the 3' primer does not have sequence similarity to GTGG.
- The molar ratio of PCR product: TOPO vector used is critical to success.

We recommend using a 1:1 to 2:1 molar ratio, starting with a 1:1 of PCR product: TOPO vector. The TOPO cloning efficiency decreases significantly if the ratio of PCR product: TOPO vector is <0.1:1 or >5:1. These results are generally obtained if too little PCR product is used (i.e., PCR product is too dilute) or if too much PCR product is used in the TOPO cloning reaction. If the yield of the PCR product has been quantitated, the concentration of the PCR product may need to be adjusted before proceeding to TOPO cloning. For pENTR TOPO vectors, using 1-5 ng of a 1 kb PCR product or 5-10 ng of a 2 kb PCR product in a TOPO cloning reaction generally results in a suitable number of colonies.

I am searching for a cloning vector that has gateway compatible att sites present in the vector for later transfer to an expression vector. A directional cloning bias would be a huge advantage. Do you have any suggestions?

We would suggest using one of our pENTR D-TOPO kits for easy TOPO cloning of your insert with the advantage of directionality.