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.