| | Re: PCR with Degenerate primers
Is the set of degenerate primers tested on a group of your bacteria or just on type/reference strain(s)? I actually mean that the justification for using degenerate primers is some polymorphism within the particular gene so that standard primers won't cover all possible versions of the gene. But maybe there is some kind of possibility to design standard primers on the basis of several sequences of the gene from several diferent strains? This will require some sequencing though.
This is not an easy issue to solve. If you have no other options and there is some polymorphism in the sequence you study then you'll need to be sure that your degenerate primers are specific enough. I would recommend thorough specificity test on a number of different templates (not only on DNA from closely related bacteria). There is GenBank and blast, but some genomes are not fully known yet...
It doesn't matter if you fall down as long as you pick something up from the floor when you get up.