Excellent Reagent Mix From New England Labs for the Assembly of Large DNA Plasmids

We study how different resistance mutations can affect Dengue virus replication. To do that, we need to obtain viral genetic material containing the desired mutation. This reaction mix allows an easy and fast way to generate full-length cDNA infectious clones. First, we double-digested a wild-type Dengue virus infectious clone and recovered the plasmid backbone. Then, we designed primers with overlapping regions containing the mutation of interest and performed PCR to introduce it into the infectious clone. The fragments generated can be connected to the plasmid backbone in a single reaction and generate the mutant Dengue infectious clone (around 14 kb). In my case, I had three fragments to assemble (2 overlapping fragments and 1 plasmid backbone) so I used the following ratio: 1:2:2, which corresponded to 50 ng of plasmid backbone + 100 ng of each fragment. In a 20 uL reaction, I added 10 uL of the NEB assembly master mix and completed the volume with deionized water. I incubated the reaction for 25 minutes at 50 °C and proceeded to the transformation of competent E. coli EPI300 cells by heat shock using the entire reaction volume. After a 16h-incubation I obtained around 50 colonies. Five colonies were picked to be sequenced, and all five were confirmed. This reaction mix is frequently used in my lab for its convenience since we can use PCR products directly in the reaction and there is no need for additional steps with restriction enzymes (like a DNA ligase reaction would require).