Preparation of bacteriophage and plasmid DNA.

Transfection of F' E. coli cells with the Ligation Mixture of topA DNA Inserted into M13mp19.

---

In the last period we subcloned the gel purified, blunt-end PCR product containing 5'-phosphates into the phage vector, M13mp19. The insert will be cloned (hopefully) in either orientation. Why?

---

Following transfection, the E. coli cells will produce phage particles that contain single-stranded DNA (+ strand). This DNA will contain one strand of the PCR product

or the other.

---

After transfection and plating, turbid plaques are produced on the lawn of E. coli cells.

---

Color screening will allow us to identify phage bearing an insert from phage with no insert.

---

We will propagate phage from "white" plaques in the next lab period and then determine the orientation of the insert in Experiment 13.

---