A geneticist aims to insert a eukaryotic gene into a plasmid vector containing an ampicillin resistance gene (AmpRAmp^RAmpR) and a tetracycline resistance gene (TetRTet^RTetR). The restriction site used for the insertion lies within the TetR Tet^R\,TetR gene, leading to its inactivation in recombinant plasmids.
Which of the following options shows the correct sequence of procedures to successfully obtain and identify bacterial colonies containing the recombinant plasmid?
Digest eukaryotic DNA and plasmid with different restriction endonucleases →\rightarrow→ mix with DNA ligase →\rightarrow→ transform bacteria →\rightarrow→ plate on agar with ampicillin →\rightarrow→ select colonies that grow on tetracycline
Digest eukaryotic DNA and plasmid with the same restriction endonuclease →\rightarrow→ mix with DNA ligase →\rightarrow→ transform bacteria →\rightarrow→ plate on agar with tetracycline →\rightarrow→ replica-plate onto agar with ampicillin and select colonies that grow on ampicillin
Digest eukaryotic DNA and plasmid with the same restriction endonuclease →\rightarrow→ mix with DNA ligase →\rightarrow→ transform bacteria →\rightarrow→ plate on agar with ampicillin →\rightarrow→ replica-plate onto agar with tetracycline and select colonies that fail to grow on tetracycline
Mix eukaryotic DNA and plasmid with DNA ligase →\rightarrow→ digest with the same restriction endonuclease →\rightarrow→ transform bacteria →\rightarrow→ plate on agar with ampicillin →\rightarrow→ replica-plate onto agar with tetracycline and select colonies that grow on tetracycline
