User:Barry Canton/Notebook/T7 RNAP transcription of rRNA/2008/08/08

From OpenWetWare
Jump to: navigation, search
<html><img src="/images/c/c3/Resultset_previous.png" border="0"/></html>Previous entry T7 RNAP transcription of rRNA Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Ok, got the first successful dot blots yesterday (images are on TK's gel imager). Making the dot blot work seemed to depend on either using a different wash buffer (6x SSC) or the fact that I switched to the chemiluminescent reagent. I'd like to figure this out since the ECF signal is supposed to be more linear than the ECL one.

Also, the fact that the dot blots work don't mean I'm specifically detecting the 16s, I might need to run an actual Northern to confirm this. I'll also run a dot blot using my pure RNA test targets. I think I

Things I need to do now -

  • Confirm that the probe is binding specifically to 16s rRNA (run Northern, and or run standard RNA dot blot)
  • Confirm that the orthogonal probe also works
  • Figure out hyb/wash conditions such that there is specific recognition of the two targets

It would be nice if I could run a gel and a transfer today. Not sure if that is possible. Might be. Dot blotting. I probably want to make multiple serial dilutions of the RNA standards and dot them. I'll need at least two if I'm going to prepare labeled probe.