9:00A – 7:30P, 8:15P – 11:15P
- Prepping gel (low on Na Borax, and TAE making up new TAE)
- Test gel: YW postive control AbdB looks great. YW negative control sim[D] is clean. Improvised gel extractions not looking good.
- Try sim[D] DNA extraction again?
- Fractionate flies in 100 uL ddH2O,
- Lyse 2hrs in 500 uL DNazol
- spin down fly parts, remove supernatant.
- Add 400 uL 100% EtOH, invert to mix. React at RT 3 min.
- Attempt to spool out DNA: no sign of spooling.
- Tried precipitating. Lost pellet in air drying.
- VWR fly homoginizer: PelletPestle
- rinsing out primary antibodies
- Block and add secondaries
- collect virgins
Draft letter to MERK. Sent to Barbara.
Probe synthesis
- kappa Taq arrived. Setting up 10 mL of PCR reactions in 96 well plates.
- Froze samples
- Wrote to Brian for clarifications on next steps.
Confocal observations
- (Fab 7 should have had K27me3 in Cy3B). almost empty tube of dk a-rb Cy3B (concentrated) looks like absolute crap — backgroundy and no K27me3 to speak of. (was 1:400, but Scm looks good at 1:400, some nuclei extremely strong). Fab7 okay but probe still backgroundy a bit. Shouldn’t have skipped most of the hot washes — but at least the probe works!
- SCM extreme variability in phenotypes. no global de-repression of AbdB. Late embryos that manage to form CNS show widespread AbdB in all the CNS and patches of AbdB at random throughout the ectoderm. Will write more detailed report in separate post.
- sim[D] — should drop probe and antibody concentrations — signal very bright but bright background spots, especially with sna — bright background foci not seen in last set of snail staining.
Discuss ultracryo with Colesno.
- bring samples
- let’s try the centromere labeled and Fab7 labeled embryos as well as some unlabeled ones.
- how small to imbed: needs to fit on tip of pin (will be frozen to bin). a few embryos in a bundle.
- Recommend embedding small cluster of embryos in a block of agarose (colenso has low MT agarose downstairs).
- Cryo protect.
- snap freeze in liquid nitrogen at facility and trim block with razor blade
- what to bring? (cleaned coverslips. Lysine coated work best).
- Contact Maria Ericsson at HMS EM facility about getting started with ultracryo.