Good news! I started my project over again, and everything is working! I isolated plasmids from BP clonase colonies, and the insert was in the plasmid, as confirmed after sequencing. The LR reaction was run after this, using both PDEST and PLEX as destination vectors so that both bacteria and mammals can express this gene. The LR reaction was also successful for both PDEST and PLEX, and plasmids were isolated for both.
I found it surprising how fast Gateway cloning was compared to traditional cloning, which didn’t work. For traditional cloning, there were two colonies on one plate, but those colonies were “fake” colonies that grew for other reasons. I think they were mutants.
The next part of my project is to make a lentivirus, so that my insert can be delivered into host cells, so it functions as a gene delivery vector. I am using HEK cells to make the lentivirus. So far, I am growing two flasks with HEK cells and I am waiting for the cells to be confluenced so that I can transfect these cells with my insert.