Wednesday 04/25/12

9:45 A – 8:30 P

  • O/N hybridization — should do in PCR block.  Water evaporates from 37C block, not good contact with PCR tube.  Refilled water, allow another hour of hybrdization before washes.
  • Sequencing results:  A1 = Taf1 antisense (sequence with m13F = T7 side) use T7 for antisense probe
  • A2 = Taf1 sense -> use  SP6 for antisense probe
  • U1 = alphaTub sense -> use  SP6 for antisense probe
  • U2 = alphaTub48b antisense (sequence with m13F).  has 500 bp homology with aphaTub84D (similar genomic location)
  • setup PCR to linearize Taf and Tub for probe making:
  • Repeat PCR for BX-C probes: Fab8, iab2E, iab7E, iab8E, AbdB2, Dfd1, try 60 annealing instead of 72C.  max probe length is 2kb, try 3 min elongation.
  • setup probe making reactions for en and hox loci.  KE didn’t label gel extracts, just ID numbers.  ID constructs by size:
  • Running test gel of BX-C cloning round 2:
    Dfd1, AbdB2, iab8E, iab7E, iab2E, Fab8 (respectively)
  • Running test gel of PCR linearization of Taf and Tub templates (respectively)

  • Running test gel of in vitro transcription reactions of en, inv, tou and Epc (respectively)
  • Make 50 mL STOP soln.   Use RNAse free H2O
  • Make 50 mL 2x Carbonate buffer.  Use RNAse free H2O
  • Blocking hybridized embryo
  • incubate with ch a-GFP, m a-Dmo, rb a-H3K27
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