This is a fine result. I'm now using 0.5 µl of the high-template reaction as the template in a new reaction with the same F and R primers and an annealing temperature optimized for them. I hope this will give me lots of the full-length product.
In anticipation of having the desired full-length DNA fragment, I've just streaked out the recipient Actinobacillus pleuropneumoniae cells I will transform this fragment into. There are several steps I need to do before the final transformation:
- Streak out the honours undergrad's A. pleuropneumoniae SpcR mutants (she made three different ones with the same cassette I'm using).
- Check the sensitivity of A. pleuropneumoniae to spectinomycin, since this is the selection I will be using for transformation by my fragment. The honours undergrad did this but her notes are not very good here. I need to identify a concentration that will prevent colony formation by the sensitive cells but allow it by the resistant cells.
- Make a competent stock of the recipient (SpcS) by growing the cells in MIV starvation medium.
- Check the competence of these cells by transforming them with genetically marked DNA. I know I have some old DNA for this purpose (NalR?), but it would be good to select for SpcR using DNA from one of the undergrad's SpcR strains, if I can find this.