pSpark®

• Selecting the right pSpark®
• Low amount DNA cloning
• Cloning of long DNA fragment
• pSpark® and your time
• Sample request by email
• Beyond Pfu

Although Taq polymerase is a familiar highly robust enzyme in every molecular biology laboratory, this enzyme introduces mutations in a large percentage of amplified molecules producing a considerable lost of time in cloning and in recombinant protein expression procedures. Second generation of high fidelity DNA polymerases is the solution to the high percentage of reaction failures, low yields and large optimisation steps that are needed with first generation proofreading polymerases, like Pfu.

Nowadays, high fidelity DNA polymerases are represented by enzymes such as Phusion®, iProof™, KAPAHiFi™ and PfuUltra™. Internal R&D at Canvax has used Phusion®, iProof™ and also KAPAHiFi™ DNA polymerases in over 1000 different amplifications over the last two years and we have found they are as robust and easy to be used as Taq polymerase and in over 800.000 bases sequenced to date we have not found any mutations; using both Taq polymerase and blends of polymerases we have found that more than 10% of sequenced clones had mutations.

The pSpark® DNA cloning systems are designed for the cloning of blunt PCR products amplified by proofreading or high fidelity DNA polymerases. Those polymerases include first generation thermostable DNA polymerases where Pfu, Pwo, KOD HiFi and Platinum® Pfx DNA polymerases are prototypes. Cloning into pSpark® can also be done directly (without a blunting step after PCR) with polymerase blends such as Expand™ High Fidelity PCR System, Platinum® Taq High Fidelity and AccuTaq® DNA polymerase but wue strongly recommend second generation thermostable high fidelity DNA polymerases like Phusion®, iProof™, KAPAHiFi™ and PfuUltra™.

To clone a DNA fragment, the first choice that you have to do is the polymerase enzyme. Selection of a polymerase for PCR depends strongly of polymerase availability in the lab but enzyme properties like the ends that the enzyme produces, accuracy, size of fragments amplified, suitability of the ends for an specific cloning vector must be highly relevant for success and productivity.

Next table lists some proofreading or high fidelity DNA polymerases that produce blunt end and could be used for pSpark® based DNA cloning. We strongly recommend second generation proofreading polymerases for cloning into pSpark® and following manufacturers instructions for using them.