Team:Wisconsin-Madison/parts 4


PFU Polymerase

Pfu polymerase is a high-fidelity polymerase originating from the hyperthermophilic organism Pyrococcus furiosus. Often used in PCR, Pfu contains 3’ to 5’ proofreading activity, making it an attractive alternative to other polymerases (Lundberg, 1991). Pfu polymerase was used in our in-house Gibson Master Mix, replacing Phusion DNA polymerase due to its similar activity as a thermostable DNA polymerase with minimal error rates. Pfu has been purified by a member of the Pfleger Lab, providing us with a knowledgeable source regarding proper enzyme purification.

The PFU gene is inserted in an ampicillin resistant PET 11a plasmid under the T7 promoter and terminator. It has a hexahistidine tag, and so its expression can be induced with IPTG and purified using Ni-NTA agarose beads.