9:10 A – 6:00P, 9:30P – 11:20 P
- start thawing DNA Fix Mix at 37C
- email BIRS team about NIH funding option update
- Need to coat new coverslips. Contacting olympus about #1.5 vs #1 coverslips. (Zeiss recommends only 1.5, strong difference in photons, especially for high mag high NA lens). Olympus confirms .17mm (#1.5)
Troubleshooting DNA labeling for embedding
- DNA not well cross-linked by formaldehyde? Wiki says makes protein-DNA and protein-protein crosslinks, reversed by heating at 70C.
- RNA-probes antibodies probably get well fixed. 20bp DNA probes don’t get properly fixed. maybe use biotin and fish with a secondary?
- On formaldehyde vs. gluteraldehyde from MicroscopyToday. FA fast must be in low concentration to be monomer to be effective. methanol added to formalin (37% formaldhyde to slow polymerization). Alternatively heat to 60C in physiological buffer.
- GA reacts protein only (JMicroscopy1984). Separates out DNA from chromatin after GA fix.
- Difference from previous stain? shelf formaldehyde quality has gone down due to polymerization? Polymerization temperature was lower? 60C setting?
- Results from test of effects of EtOH on resin
- 50 uL EtOH (in 1 mL resin) = still liquid at 15 hrs, very pliable at 18 hrs
- 15 uL EtOH + 1 mL resin pliable
- pure resin is solid.
- Need to do more pure resin rinses throughs, maybe suction for longer, really get rid of previous resin from chamber. Hopefully this also helps with etching. (need to make new sodium ethoxide solution too).
- To Test
- Check if probes disappear after EtOH or after heating.
- Try heating fix-mix to 60C briefly before use.
- ordered new dig-labeled centromere probe. Try this.
Fly Work: embyro collection
- poor O/N egg lay… maybe not worth fixing…
- Flip collection cage plate at 11:00 A.
- Heat DNA fix mix up to 60C (break PFA into monomers). Fixing 1-13 hr embryos for DNA-FISH. Stored in MeOH at -20C in Projects box.
- MTD x TRiP crosses ready to get flipped, but out of new bottles. Should flip later this week (when/if?) new bottles arrive.
- Flip collection cage, 9:50 PM
- Check Su(Z)12 6-15 staining (SuZ + Dm01. Not visually evident SuZ…).
- Fix embryos: 10:50 PM. Rewarm to 60C the D-FISH Fix mix.
- thaw some fresh Pc antibody. Don’t have time to block and stain -> do so with tomorrow night’s embryos. Just fix these and have some more wt (MTD) to stain.
New antibodies labeled:
- AlexaDyes data for labeling reactions
- dk a-shp IR-800 (extinction coeff: 240,000; 280nm CF at 280: 0.03). added 2.5 uL of 5 mg/mL solution. 1 dye/AB
- dk a-shp A514 ext. coef: 80,000 CF at 280: 0.18. Diluted to 2 mg/mL, added 2.5 uL: 6 dyes/AB
- dk a-shp A534 ext. coef: 81,000 CF at 280: 0.09 Diluted to 2 mg/mL, added 2.5 uL: 3 dyes/AB
Probe making for DNA FISH
- Talk to John about nicked translation protocol.
- Ordered more separate dNTPs (need to adjust labeled to unlabeled ratio of Ts).
- DNA probe synthesis for BAC7.
- 5 uL DNAse working solution (.66 uL in 100 uL buffer).
- 1.7 uL dUTP-Cy5 (25 nmol in ~25 uL) 1 mM. (JW recommends 4uL of .2 mM).
- incubating for 2hrs at 16C (followed by heat denaturation 15min 75C, then 4C)
- Screwed up first round (2 min not 2 hr incubation!)
- Set up new round of Nicked translation.
- Also added new enzyme to previous batch and reset.
- Probe in -20C in EtOH O/N (spin precipitate tomorrow).