The whole workflow, one mission: get a gene into a vector, ready for its organism. Pick enzymes, run PCR, verify it survived, then splice. Every step is real.
Pick a pair that cuts the vector's MCS (so the insert can go in) — but beware: an enzyme that also cuts inside the gene will shred your insert. You won't know for sure until the gel. Two different enzymes give a directional clone.
③ PCR — amplify the gene & add the sites
Your primers anneal to the gene's ends and carry 5'-tails (amber) that stamp the restriction sites onto the product.
④ Verify — did the insert survive?
Diagnostic digest of your PCR product with both enzymes. A clean insert drops out as one band at the gene's size. If an enzyme cut inside the gene, you'll see it split into extra bands — the insert is ruined.
Running the gel…
⑤ Execute the splice
⑥ Confirm the construct
Final diagnostic digest of the recombinant plasmid: backbone + insert, exactly as designed.