Thermo Scientific DNA polymerases and cloning

Thermo Scientific Phusion® DNA polymerases  have an extremely low error rate, which makes them the ideal choice for cloning. Therefore we strongly recommend Phusion DNA polymerases for all cloning applications. Below you can find information about cloning PCR products amplified with Phusion.

However, if you are cloning PCR products amplified with other Thermo Scientific DNA polymerases, you need to consider that different DNA polymerases create different type of ends in the PCR products, which affects the subsequent cloning procedure. Additional information can be found below.

Phusion DNA polymerases – the best choice for high-fidelity cloning

Phusion® DNA Polymerases create blunt end DNA products. When cloning fragments amplified with Phusion DNA Polymerases, blunt end cloning is recommended.

If TA cloning is required, it can be performed by adding 3' A overhangs to the blunt PCR product with a different polymerase (e.g. Taq DNA Polymerase).

1. Purify the PCR product (e.g. with a PCR purification kit or phenol extraction and DNA precipitation)
Before adding the overhangs it is very important to remove all the Phusion DNA Polymerase by purifying the PCR product carefully, as the proofreading activity in Phusion DNA Polymerase is very strong. Any remaining Phusion DNA Polymerase will degrade the A overhangs, thus creating blunt ends again.

2. A-addition with Taq DNA Polymerase

Reaction components:

• Purified PCR product
• 0.2 mM dATP
• 1x Optimized DyNAzyme™ Buffer
• 1 U DyNAzyme II DNA Polymerase

Incubate 20 min at 72 °C.

3. Proceed to TA cloning. For optimal ligation efficiency, we recommend using fresh PCR products, since 3´A-overhangs will gradually be lost during storage.