Cloning into dual expression vector pTH
There are two main ways we recommend cloning into plasmid pTH.
1. Digest the vector with I-SceI and CeuI to open up two cloning sites, precipitate both fragments,
combine with amplified inserts that contain appropriate homology to one or the other cloning
site, perform Gibson assembly to assemble the four pieces into one plasmid.
2. Amplify the two vector fragments (inside and outside the I-SceI and CeuI sites), combine with
amplified inserts that contain appropriate homology to one or the other cloning site, perform
Gibson assembly to assemble the four pieces into one plasmid.
While the second approach is slightly more work, it has been somewhat more effective in our hands.