PfxUltima with Zero Blunt® TOPO® PCR Cloning Kit with One Shot® TOP10 Chemically Competent E. col - FAQs

pCR2.1-TOPO和pCR4-TOPO载体有什么区别？

两个载体的骨架非常相似。主要的不同在于pCR4-TOPO载体测序引物位点距离PCR产物插入位点仅33 bp。这使得对插入序列进行测序时需要读取的载体序列能最小化，从而使pCR4-TOPO载体非常适合进行测序应用。

pCR2.1-TOPO 和pCRII-TOPO载体的区别是什么？

两个载体的骨架非常相似。主要的不同在于pCRII-TOPO 载体是一个双启动子载体，包含SP6和T7启动子用于体外转录/测序，而 pCR2.1-TOPO仅包含T7启动子用于体外转录/测序。两个载体均含有M13 正向和反向引物位点用于测序或PCR检测。

你们的TOPO克隆试剂盒包含一个对照模板和一个对照引物。我可以获得对照模板的序列吗？

对照模板的序列是受专利保护的。

我可以用pCR-Blunt 或pCR-Blunt-TOPO载体克隆使用Taq DNA聚合酶扩增而来的PCR产物吗？

不，你不能用pCR-Blunt 或pCR-Blunt-TOPO载体克隆使用Taq DNA聚合酶扩增而来的PCR产物。 pCR-Blunt载体被制备为平末端以接受平末端片段。因为Taq DNA聚合酶的末端转移活性，该酶产生的PCR产物带有3’-A突起。为了将这些PCR产物克隆进pCR-Blunt，你需要将它们的末端抹平（这通常是一个低效的过程）。我们的TA Cloning试剂盒或TOPO TA Cloning试剂盒对于克隆Taq酶生成的PCR产物来说是更好的选择。 TA Cloning试剂盒包含带有3’-T突起的线性化载体用于高效连接Taq酶生成的PCR产物，无需额外操作。

What is the difference between the pCR2.1-TOPO and pCR4-TOPO vectors?

The vector backbones for both of these vectors are very similar. The main difference is that the pCR4-TOPO vector has sequencing primer sites located as close as 33 base pairs from the PCR product insertion site. This minimizes the amount of vector DNA sequence that needs to be read before reaching the sequence of the insert, making the pCR4-TOPO vector very useful for sequencing applications.

What is the difference between the pCR2.1-TOPO and pCRII-TOPO vectors?

The vector backbones for both of these vectors are very similar. The main difference is that the pCRII-TOPO vector is a dual promoter vector, containing the SP6 and T7 promoters for in vitro transcription/sequencing, whereas the pCR2.1-TOPO vector contains only the T7 promoter for in vitro transcription/sequencing. Both vectors contain the M13 Forward and Reverse primer sites for sequencing or PCR screening.

Your TOPO cloning kits contain a control template and control primers. Can I obtain the sequence of the control template?

The sequence of the control template is proprietary.

What PCR enzyme would you recommend for use with the Directional TOPO Cloning Kits?

For the Directional TOPO Cloning Vectors, a PCR product must be generated by a proofreading enzyme to create a blunt product. Pfx50 or Accuprime Pfx and Accuprime Pfx Supermix from Thermo Fisher Scientific are recommended for use.

When cloning a Pfx-amplified PCR product, the insert to vector ratio is an important consideration. The PCR product generally needs to be diluted since Pfx generates a high concentration of product and using too much insert DNA can hamper the TOPO reaction. A 1:1 molar ratio of vector to insert (or about 2-10ng of insert) is recommended.

Can I use the pCR-Blunt or pCR-Blunt-TOPO vector to clone PCR products amplified with Taq DNA polymerase?

No you cannot use pCR-Blunt or pCR-Blunt-TOPO vector to clone PCR products amplified with Taq DNA polymerase. The pCR-Blunt vector is prepared with blunt ends to accept blunt-ended fragments. Due to the terminal transferase activity of Taq DNA polymerase, PCR products amplified with this enzyme have 3'-A overhangs. In order to clone these products into pCR-Blunt, you would need to polish the ends to make them blunt (which usually is not an efficient process). Our TA Cloning Kits or TOPO TA Cloning kits are a better choice for cloning Taq-generated PCR products. TA Cloning kits include a linearized vector with 3'-T overhangs for efficient ligation of Taq-generated PCR products without additional manipulation.

What are the melting temperatures for the M13 Forward (-20) and M13 Reverse primers in the TOPO Cloning and Zero Blunt Kits?

Assuming that the primer is at a 50 nM final concentration and 50 mM final salt concentration, the melting temperatures are: M13 Forward (-20) Primer = 52.7 and the M13 Reverse Primer = 45.3. For use in the control PCR reaction we recommend using an annealing temperature of 56C.