Semi-success

Here are the results of the first attempt at getting full-length PCR products:

In the left lane (high template) I see a faint full-length band, and a stronger band the expected size of one of the expected intermediates.  In the right lane (low template) I see only the intermediate band.

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.

Before doing the final transformation I should also digest my transforming fragment with a couple of diagnostic restriction enzymes, just to be sure it is what I want.