Learning Module - Experiment 11C

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?

Insert can be ligated in either orientation

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

Orientation A inserted into phage

or the other.

Orientation B inserted into phage

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

Plating a single plaque

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

Color screening for recombinants

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

Inserts may be from either strand