Learning Module - Experiment 9A

Amplification of topA DNA using the Polymerase Chain Reaction

The DNA template we will use is the pTrc99A/topAcysB plasmid. It is combined with the two PCR primers, dNTPs and thermostable DNA polymerase. The mixture is heated to 94° C to induce strand separation, then cooled to 65° C to allow annealing of the primers. Then the primers are elongated by incubation at 72° C.

PCR amplification, primer annealing

The heating, cooling, elongation cycle is repeated:

PCR amplification, extension

In the next cycle, the strands are then separated .....

PCR amplification, strand separation

..... the primers are annealed and elongated:

PCR products

The desired PCR product will continue to increase exponentially with each cycle, while the other products continue to increase arithmatically.

When template DNA is omitted, no PCR product is formed. A variety of different PCR products are formed when using only one primer in the presence of template.

Gel showing PCR products

The PCR product is then purified by column chromatography for cloning into M13mp19.