10:15 am – 7:30 pm
Goals
- Finish RNA hybes and check hb RNA stains
- Finish embryo BX-C 15 kb region hybes and check stains
- Finish draft of ppt presentation for pairing meeting.
Probe synthesis
- Mix:
- 20 uL RNA
- 8 uL 5x buffer
- 4 uL primer (P2 – P6)
- 3 uL dNTPs
- 2 uL Maxima
- 3 uL RNasin
- total 40 uL
- Master Mix 9x
- 72 uL buffer
- 27 uL dNTPs
- 18 uL Maxima
- 27 uL RNasin
- incubate 1 hr at 50C
- digest RNA with 50% 8mM NaOH + 50% .5M EDTA pH 8.0
- P order: C01 to C08 are P2, P4, P5, P6, P2, P4, P5, P6
- unfortunately the large bands appear to be stable concatimers in the ssDNA gel:
- I bet they still work as probes, but it would be better if they came out the designed length.
- I’m not sure what this does to my probe diversity
Embryo staining
- imaging samples on STORM5
- made new settings files in data/Alistair/settings on STORM5
- bright stains on E20-E24
- looks like decent staining on E24 alone. Will be easier to tell with mosaic tiling,
- hb-RNA looks reasonable, sections are a bit thick for easy smFISH, single fluors overlap.
- also don’t have any nice cross-sections of early embryos, which would be best for analysis.
- still looks promising. next time try at lower concentration.
RNAi
- finish dsRNA synthesis
- digest DNA with DNase I + 30 uL ddH2O dilution (15 min at 37C)
- purify RNA with RNA XP beads, eluted in 200 uL. Ph-p at ~1000 ng/uL and Ph-d at ~1500 ng/uL (this difference in production efficiency is quite reliable…)
- 30 uL Ph-p and 40 Ph-d
- 50 min treatment, then added serum