Workflow notes and controls

Workflow, in order:

1. Cut: A restriction enzyme (for example, EcoRI) cuts both the human gene source and the plasmid vector at the same recognition site.

2. Ligate: DNA ligase seals the insert into the cut plasmid, joining the sugar-phosphate backbone.

3. Transform: Competent bacterial cells take up the recombinant plasmid during heat shock.

4. Select: Cells are plated on antibiotic agar so only those carrying the resistance plasmid survive.

Why restriction enzymes cut at specific sequences: They recognize a specific short palindromic sequence and cut only there, producing matching sticky ends so the insert and vector fit together predictably.

Controls:

• Positive control: cells given a plasmid known to carry the resistance gene; they should grow on antibiotic agar, confirming the transformation and plates worked.
• Negative control: cells given no plasmid, plated on antibiotic agar; they should NOT grow. If they do, the antibiotic failed or the plate is contaminated.

Safety reason for controls: The negative control catches contamination and confirms the antibiotic is actually killing non-transformed cells, so we do not mistakenly release or misidentify untreated bacteria.