Efficient High-fidelity DNA Polymerase From Invitrogen for Site-Directed Mutagenesis

We study how certain mutations affect viral replication. We construct our mutant cDNA infectious clones by PCR and this DNA polymerase mx is extremely convenient to this end. In this case, my reaction consisted of mixing my template DNA (a Dengue cDNA infectious clone around 14 kb), mutagenic overlapping primers, the reaction mix, and deionized water. The polymerase mix contains everything necessary for the reaction, which saves time and reduces the chances of contamination. Since my template DNA is quite long, I usually use 30-50 ng of template DNA, an elongation time of 7 minutes, and a final extension of 10 minutes. Then, it is necessary to treat your PCR product with DpnI to remove your template DNA (I usually add 1 uL of enzyme directly to a 50 uL reaction and incubate at 37 °C for 1h). Finally, DNA is ready to be transformed into bacterial cells. This polymerase possesses high fidelity and processivity, which is essential when doing this kind of experiment to ensure unwanted modifications to your sequence.