Saturday 10/12/12

10:30 A – 7:00P,

Experiment planning stuff

DNA FISH

centromere labeling
test different post-fix conditions and check staining at different post-fix steps to ID loss of DNA-FISH labeling.
Incubating in probe, ~4:00P

Snail in situs

Multiplex RNA-FISH+STORM

Pc + hox + en
H3K27 + hox + en with new 514 / 534 probes
rehydrated embryos first from MeOH, then dehydrated back in EtOH for xylenes clearing.
[spreadsheet 0AjSqkxgziU1YdG90a29UaU5ua1FLUXZ5LTFmM1pMSlE 602 302 sheet=8]
Incubating in probe, 6:45 PM

Literature:

McGinnis also sees ‘spurious’ Antp transcription in posterior cells (PLoSONE), along with maintained expression in the ventral nerve chord of segments T2 – A7 of stage14 embryos. Coexpressed in A3 to A8 with iab-4 from BX-C (though rest of BX-C is Pc silenced presumably).
Agelopoulos: pull down promoter DNA from Active cells only (using LacI bound to LacO sites there, LacI is only expressed in the active cells) find all the enhancers. Pull down promoter DNA in repressed cells, only promoter proximal region is found. (Also indicates repression is by anti-looping of repressive ‘304-element’ (12Kb 5′)).
CR43617 is probe ‘6’ in Bae/Calhoun/Levine paper. Has both sense and anti-sense transcription. iab6 side of Fab-7. antisense transcription looks to make mature transcripts. Pattern is complimentary to sense pattern (sorta. stronger more anterior and weaker more posterior).
Pretty sure different anterior boundaries of different iab regions in Bae paper is BS based on my double in situs. (plus the fact its all one iab8 transcript)

# Probes to transcribe