Monday: PCR controls show negative result
The next step: Actually, thinking about it, I'm not so bad off and I am not starting over. I just now have to tweak my system. I am suspecting that rtcB is not ligating in this case because the splint is DNA and it might not like ligating RNA/DNA duplexes. I'm going to try an RNA splint and see where that takes me.
Tuesday: Is rtcB ligating?
Result: I did see ligation - but not much - it was less than 10%. I'm optimizing the reaction conditions the rest of this week to see if I can increase that. Wish me luck!