- Starting with a circular plasmid containing the short 'Toxin' and 'Antitoxin' genes, I'll use inverse PCR to amplify a linear fragment that lacks most of the coding sequences of the Antitoxin gene.
- I'll also amplify (or find) a SpcR cassette.
- I'll ligate these two molecules together to create a circular plasmid with the SpcR cassette replacing the Antitoxin gene. I could do this by blunt-end ligation (what the Honours student originally did) or do it as the student originally planned, using conventional ligation of 'sticky ends' generated by digesting both fragments with a restriction enzyme whose site is present at all the ends (she designed it into the primers). I think she changed her plan because our stock of this enzyme was inactive.
- I'll use this plasmid (linearized by cutting somewhere in the vector) to transform the bacterium Actinobacillus pleuropneumoniae to SpcR.
- I'll use PCR of chromosomal DNA from the new resistant transformant to check that the original antitoxin gene has been replaced by the SpcR cassette.
- Next I'll do a transformation assay on this mutant to see if its competence has changed, with the wildtype and toxin mutant as positive controls.
Step 1 actually requires that I get off my butt and:
- Resuspend the primers at the appropriate concentration of TE (or water?). (Check with the grad student.)
- Find the PCR reagents. (Ask the grad student).
- Find the template. (Find info provided by Honours student before she left.)
- Learn how to run the PCR machine (Ask the grad student.)
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