A cloning enzyme has to pass three checks
- The idea
- Predict
- See it
- Why
- Check
The idea
“If an enzyme opens the plasmid where the gene goes, it's the one to use.”
Once an enzyme opens the plasmid in the right place, it feels settled. It can still wreck the gene or the marker.
The short version
The mistake: If an enzyme opens the plasmid where the gene goes, it's the one to use.
What's actually true: A cloning enzyme must open the plasmid at the cloning site, leave the gene whole, and leave the resistance gene uncut.
- A restriction enzyme recognizes one short DNA sequence and cuts wherever it appears. So check every place it cuts, in the gene and in the plasmid.
- The gene has to stay in one piece. A cut inside it leaves fragments with only part of the coding sequence.
- The resistance gene is the selectable marker. Only cells with a working copy survive the antibiotic. That's how you find cells holding the plasmid.
On the exam: Check every enzyme three ways: opens the plasmid, spares the gene, spares the marker.