HomeFAQsCan I use Q5U Hot Start High-Fidelity DNA Polymerase for USER® cloning methods?
FAQ: Can I use Q5U Hot Start High-Fidelity DNA Polymerase for USER® cloning methods?
Yes. Q5U Hot Start High-Fidelity DNA Polymerase is a modified Q5 High-Fidelity DNA polymerase which efficiently amplifies uracil-containing templates and primers. For USER cloning methods, target DNA molecules and cloning vector are generated by PCR with 8-12 bases of homology between two fragments. PCR primers start with a 5′ A and contain a single deoxyuracil residue (dU) flanking the 3′ end of the homology region, and can be designed to accommodate multiple-fragment assembly, nucleotide substitutions, insertions and/or deletions. We recommend using the GeneDesign software to design primers for USER junctions. The best results are typically seen when using each primer at a final concentration of 0.5 µM. In comparison to Taq-based enzymes, Q5U Hot Start High-Fidelity DNA Polymerase makes significantly fewer errors, streamlining any cloning protocol (for example, see data below)