9:30 A – 7:30 P
- Flip collection plate on MTD flies. starting to lay…. sorta.
- looking at aptamers for histone labeling (see Mendeley).
- DNA-FISH + protein prep for STORM protocol test try 2: samples now embedding.
Trends suggest current donkey anti-rabbit is no good :(. Better switch soon.
Datasets in review:
- ON Alistair6
- 2012-08-16_AA: good used for temporal analysis of K27me3 accumulation at AbdB locus in AbdB silent cells.
- 2012-08-17_Dfd: good. Not cluster analyzed.
- 2012-08-25_AbdA: bad. 647 bleached by high 405. Quadview channel misalignment serious.
- 2012-08-28_Pc6: bad, 647 bleached by high 405.
- 2012-08-29_K27Pc: poor: low labeling density (see more comments below). see data
- 2012-08-30: good, analyzed see post.
- 2012-08-31_Pc6: scope issues, only calibration data set.
- 2012-09-06_en: small dataset (3cals, 3storms), good images, good age choices, few en loci
- 2012-09-10_s6: (5E-dig, AdbAbio). good. used for temporal analysis of K27me3 at AbdB in AbdB expressing cells.
On Alistair5
- 2012-08-02_ubx-AB. Not analyzed. Poor acquisition gain parameters
- 2012-08-04_ubx. okay images (despite acquisition parameter issue). few localcizations. good channel alignment, nice H3 (bit sparse but usable), nice K27me3, good overlap with ubx.
- 2012-08-05-tub-no647storm (forgot to open shutter for STORM images)
- 2012-08-07_tub. Good. Used for K27me3 temporal analysis. tub marker not so hot for locus ID.
- 2012-08-13_Antp. Good. Not cluster analyzed. Not all dots look like Antp pattern (ubx jump? background?)
- 2012-09-03_Pc647. Pc-647 stain looks good (much better than in K27Pc). Pc-GFP 750 not as strong in conv or STORM (may be antibody/dye issue). Problem: with polycomb this dense, it is going to be hard to determine occupancy using probes against regions several kb to one side. Really want to look for direct overlap – the occupied space is sparse, but the 100nm adjacent region is not as sparse. Analyzed. see data.
- 2012-09-12_Epc. okay k27/H3. smallish dataset (long movies though). some etching issue–pocking of sample due to water in ethoxide.
Stupid alphanumeric ordering makes age scoring much more difficult, especially since matlab dir and windows file browser have different ways of doing alpha-numeric sort.
Analysis of K27me3, Pc double stain data:
- Overall, weak labeling of k27me3 and Pc.
- Only weak difference detectable between blastoderm and 12hr embryo in conventional image.
- H3 makes a much better counterstain than lamina. At the very least put H3 in 488 and just use conventional images