Scientific basis for the project

Students are provided with two plasmids, labelled 1 and 2: one of these is pTOPO (containing a kanamycin resistance cassette) and the other is pGEX (containing an ampicillin resistance cassette). They are asked to identify which is which using redundant approaches: bacterial transformation, diagnostic restriction digest, and PCR. Before they start their experiments, students make predictions about what the results will look like for pTOPO and pGEX and then compare the results for p1 and p2 to those predictions. They are also asked to design primers to amplify each resistance cassette.

It would be good to have more samples for each experiment so that students can integrate more controls, but we haven’t been able to do that due to the size of our PCR machines, etc.

Brief summary of each session

1. Planning session: brief presentation followed by planning and prediction of results, in pairs. Plans are signed off before students leave.
2. Transformation and PCR
3. Electrophoresis of PCR products, repeat of transformation if necessary, restriction digests
4. Electrophoresis of restriction digests. Scientific writing exercises are provided while the gel runs.

(Could be squeezed into three sessions.)

Learning objectives

By the end of this project, a student should be able to:

• Use plasmid maps to design restriction digests
• Plan experiments including appropriate positive and negative controls
• Design PCR primers to amplify a given sequence
• Predict experimental results and compare actual results to the predictions

If you decide to use this practical, please let us know about it by filling in this short form – it’s not a requirement but we’d love to hear how our ideas are being used!