What steps would you take in order to produce a recombinant DNA molecule containing a piece of human DNA that can be easily propagated in bacterial cells?

Question

What will be an ideal response?

Answer 1

Answer:
1) Cut a cloning vector (such as the plasmid pUC19) and human DNA with the same restriction enzyme (such as EcoR1 which has one cut site in pUC19).
2) Mix digested cloning vector and digested human DNA together and add ligase to covalently link the complementary overlapping (sticky) ends of the DNA molecules together.
3) Add the ligated DNA to a bacterial culture (such as E. coli) and treat the cells to encourage uptake of the DNA.
4) Spread the culture on agar plates to separate individual cells and incubate overnight to allow colonies to develop. The agar plates usually contain an antibiotic so that only cells containing the plasmid will survive and form colonies.
5) Identify which colonies contain recombinant DNA molecules (versus those that contain a plasmid without inserted human DNA) based on a screen. (Note: Students could explain the lacZ screen of blue/white colonies if that was covered or expected of students.)
6) Further screen colonies to identify genes of interest.