I realized yesterday while staring at yet more totally un-interpretable results that something must be not quite right. I went back through my notes and eventually, after some detective work I realized that I made a mistake. Months ago. A fundamental mistake. I mis-ordered two primers, one contains a stop codon, one does not (for making C-terminal fusions to my favorite protein). I copied and pasted the wrong sequence to the wrong primer name... and so I made all my constructs backwards. All the N terminal constructs aren't in frame with the tag and the C terminal ones are not fusions at all - they're untagged since the stop is intact in my protein. Crap.
This is big. I mean, it's easy to fix. I've already re-run the PCRs and will have the cloning completely re-done by the end of next week, but it means that all the experiments I did (and all the transgenics I made) since, oh, say, January or so have been completely and totally useless. I am so pissed at myself. How could I have made such a stupid mistake? And not noticed it? Yes, I checked all the sequences before using the constructs. They aligned perfectly with the vector files I had made, because I also made the vector files with the mixed up primer sequences. (D'Oh!)
I'm so embarrassed. And now I feel like trying to cover my tracks and hope no one finds out... that could be difficult seeing as I now have only 15 weeks left before the kick me completely out of work and I have to re-do about 20 weeks worth of work... of course at least this time around maybe things will actually make sense and won't have to be repeated 10 times before I can get some sort of interpretable result. This was not the brightest shiniest day in the history of my scientific career.... on the bright side at least it's me that found the error and not some poor project successor that got handed a bunch of reagents that don't work.
There go my plans for a relaxing weekend break from the lab....