How do I convert pTRIPZ into Tet-Off configuration?
1- Digest pTRIPZ (with hairpin, not the empty vector) with BamHI, isolate the large vector band, and re-ligate. This will remove the rtTA3 portion of the plasmid. You can then use pTRIPZ in a co-transfection with a tTA expressing plasmid.
2- Perform a Cre/lox reaction (you can buy this from Clontech and perform the reaction in vitro, cat. number 631614). This will remove the rtTA3 portion of the pTRIPZ plasmid. Again, you can then use pTRIPZ in a co-transfection with a tTA expressing plasmid.
3- Digest pTRIPZ with BamHI and isolate the large vector band. Ligate the cut vector with a tTA fragment engineered with BamHI ends. This will replace the rtTA3 with tTA. The insert will have to be tested for proper orientation. This will essentially recreate the pTRIPZ vector only with the Tet-OFF workings rather than Tet-On. In this option, there would be no need for co-transfection as all the machinery is on one plasmid just as it was originally in the Tet-On configuration.