Thursday, 06/18/15

9:30 am – 10:45 pm

To do

  • submit progress report to DR. (done)
  • email Dell about RAM (done. Dell is very slow with reply. Probably should just go Amazon)
  • email DA about letter (with CV) (done)
  • work on slides (postponed)
  • work on DF application (postponed)

Embryo fixation

  • flip plates, 9:15 am.
  • flip plates, 1:45 pm.
  • Fix embryos 6:45 pm (6 – 9.5 hrs)

Fly RNAi

  • PCR cleanups (done).
  • order qPCR primers
  • order qPCR kit (done)
  • set up T7 reactions (done)
  • clean up dsRNA
    • out of columns
    • washed used columns with water and requilibrated with RNA binding buffer
    • this protocol is much slower — maybe I want to go back to magnetic beads…
  • set up RNAi experiments (can’t do)
  • run gels for 8 new samples (postponed)

RNA synthesis purification failed

  • attempted on-column DNase treatment following Zymo instructions (in ~70% ethanol, this is a stupid idea).
  • next time just add DNase to the in vitro mix at the end of the in vitro reaction.
  • samples seem to have degraded and run very funny on agarose borax gel.
  • testing now pre and post column purification treated RNA on a TBE acrylimide gel.
  • borax doesn’t run RNA very well, looks much better on a PAGE gel (though still awfully smeary).
  • still — T7 seems to have performed very badly, quite smeary and low yield (50-200 ng/uL in 120 uL elutions).
  • oddly sample 2 — weakest band, least smeary, has easily the highest concentration (~800 ng/uL)
  • not sure what this ‘trapped in the well’ phenomena is either…

Start again!

  • set up PCRs to amplify targets from genomic DNA and add T7
  • running PCR overnight.
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