User:Barry Canton/Notebook/T7 RNAP transcription of rRNA/2008/08/12
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Couple of things that might explain the weak signal - 1. The synthesized target sticks poorly to the membrane (only Us stick to the nylon membrane and there aren't many of them in a 30mer). 2. I had to use old labelling reagent because the current batch ran out. The kits are only supposed to be good for 3 months and I had to use 6 month old stuff.
I'm rerunning the expt. at the moment using longer target-membrane crosslinking time and fresh reagent. I also added more target to the membrane just for the hell of it.
I'm surprised that I seems to be picking up the wt target better than the orthogonal probe. In the new experiment I made fresh dilutions of the probes so there is no possibility that I accidentally used the wt probe instead of the orthogonal probe.
I also remade all the dilutions of target and probe in case I accidentally managed to mess up.