I think I finally have the appropriate PCR fragments from my A. pleuropneumoniae mutants, to be sent for sequencing:
I have 3 knockout mutants, removing the toxin, antitoxin and toxin+antitoxin segments (∆T, ∆A, and ∆TA respectively). I designed new 'S-up' and 'S-dn' primers to use with the original 'F' and 'R' primers amplify the segments on either side of the Spectinomycin-resistance cassette that's inserted at the sites of deletion. I need to check the sequences of these to be sure that the appropriate segments have been removed, and that the remaining gene is intact.
I've successfully used these primers (black arrows above) to amplify the ∆T and ∆A segments shown above (light blue and lilac bars). Now I just need to clean up the PCR products, check their concentrations, and send them with the appropriate S-up and S-dn primers (red arrows above) for sequencing. I don't need to sequence the far ends of the fragments.
I also tried to use these primers for the ∆TA double knockout but for some reason I can't get any amplification. This may mean that there's something wrong with the mutant, but I've decided I don't really need to discuss this mutant at all in our paper, since both the ∆T and ∆A mutants have normal growth and competence phenotypes. (Well, I think I do need to do at least one more check of the transformation frequencies, since there's been a lot of variation in my colony counts.)
[Ooh, idea! Maybe the ∆TA mutant won't amplify because its Spec cassette is inserted in the opposite orientation to the others! The Honours student created each mutant by blunt-end ligation, so either orientation is possible. I'll go set up one more pair of PCR reactions with the alternate combinations of primers right now...
And YES! Reversing the primers gave the expected amplification!