PCR Enzyme Selection Kit—High Fidelity - FAQs

我可以使用包含Taq聚合酶和一个校对活性聚合酶的DNA聚合酶混合物进行TOPO TA克隆吗？

如果你想使用包含Taq聚合酶和一个校对活性聚合酶的DNA聚合酶混合物，Taq酶和校对活性聚合酶的比例必须超过10:1以确保PCR产物带有3´ A突起。如果你使用的聚合酶混合物没有足够的Taq聚合酶或仅含有校对活性聚合酶，你可以在PCR反应后添加3´ A突起。请参阅产品手册获取细节。 适合TOPO TA克隆的一些Taq酶混合物的例子包括Platinum Taq DNA Polymerase High Fidelity和AccuPrime Taq DNA Polymerase High Fidelity。

Can I use a DNA polymerase mixture containing both Taq polymerase and a proofreading polymerase for TA Cloning?

If you wish to use a polymerase mixture containing Taq polymerase and a proofreading polymerase, Taq must be used in excess with a 10:1 ratio of Taq to the proofreading enzyme to ensure the presence of 3´ A-overhangs on the PCR product. If you use polymerase mixtures that do not have enough Taq polymerase or a proofreading polymerase only, you can add 3' A-overhangs following PCR. See the vector product manuals for details.

Some examples of Taq blends that are compatible with TOPO TA Cloning are Platinum Taq DNA Polymerase High Fidelity and AccuPrime Taq DNA Polymerase High Fidelity.

How does TA Cloning work?

Taq polymerase has a non-template-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3´ ends of PCR products. The linearized vector supplied in our TA Cloning kits have single, overhanging 3´ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.

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.