11:00 am – 6:30 pm
embryo smFISH optimization
- testing quality of methanol stored embryos
- from yesterday:
- 6 mo and 2 year embryo batches stained with my in situ protocol I optimized in the Levine lab
- 6 mo sample directly labelled with Stellaris FISH probes against hb.
- hot washes
- protocol with dig-probes: 3x wash 30 min each at 55, in hybe buffer.
- following Xu et al: 3x wash, 30 min each at 30C, 2x 10 min each in 2x SSCT
- no Roche western blocking soln in stock, will have to use BSA.
Testing sample
- took some confocal images of the Xu Hb-647 probes, with narrow pinhole and some zoom, single molecule images look detectable
- the antialiasing built into this system is unfortunate.
- saved some embryos to test in cryo section. Should schedule some sectioning time later this week.
- lets try these systems on our scopes tomorrow.
Probe making with Hazen
My L7 probes:
- mix: 80 uL reactions, 30 uL RNA, 16 uL buffer, 8 uL dNTPs, 6 uL 100 uM primer, 8 uL Maxima, 8 uL RNasin.
- did both newly arrived labeled and unlabeled primers
- Toe-hold probes arrived today, placed in my -20C. Bogdan will send layout of probe/well by email
HB collaborative project
- set up RT reaction in 40 uL.
- separate reactions for cy3 labeled P2 and 405 labeled P4. (should make sure Hazen has these IDs).
- Ran through RNA degradation step. didn’t get to gel — will save for tomorrow.
- wrapped gel in plastic wrap and put at 4C, turned off water bath, will run tomorrow.
Discussion with BB on setup for manual seq imaging
- Readout buffer: at RT, 40% formamide, 10% dextran sulfate, in 2x SSC + 1:1000 of 100 uM readout probe, 20-25 min incubation, use 150 uL
- 30% formamide in 2x SSC 5-10 min, 400 uL
- steven’s secondaries are in green box in downstairs -20C next to my probe template plasmids.
- im buffer as in MERFISH, keep in 4C, 200 uL to image. (Glox under oil).
To order
- I’m a bit short on some of the reagents for an ideal test.
- ordered new Roche western block, ribonucleoside vanadyl complex (RVC), RNase free BSA,
- discuss with BB ordering for new fluidics system.