Phusion™ Site-Directed Mutagenesis Kit with DH10B Competent Cells

Phusion DNA Polymerases generate blunt end products; therefore, blunt end cloning is recommended. If TA cloning is required, it can be performed by adding A overhangs to the blunt PCR product with e.g. Taq DNA Polymerase (Cat. No. EP0401). However, 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.

Yes it is possible, especially when amplifying smaller amplicons. Processivity of Phusion DNA Polymerases is 10 times that of Pfu. We recommend extension times of 15 s/kb for Phusion Flash PCR Master Mix. 15 s/kb is a conservative value that we can promise to work with almost any amplicon. In many cases, significantly shorter extension times (0-5 s/kb) can be used without compromising the yield. What separates Phusion Flash DNA Polymerase from other fast polymerases is that all steps in the PCR protocol can be shortened, including annealing and denaturation. This results in extremely fast protocols as compared with any other polymerase.

To achieve the highest possible transformation efficiency with Thermo Scientific DH5α (Cat. No. EC0112) and DH10B (Cat. No. EC0113) competent cells, please follow the transformation protocol provided with the product and pay attention to the following details:

- Make sure to use wet well-pressed ice to maximize cold surface volume in all the transformation steps.
- Perform the steps quickly and accurately.
- Use chilled microcentrifuge tubes and bury the whole tube up to the cap in the wet ice.
- Do not exceed the recommended heat shock time and transfer the tube with cells back to the wet ice immediately.