Alistair's Lab Notebook

6:00 pm – 7:00 pm

Embryo staining

• just going for labeling — set heat block to 100C
• spec new library:
• label just new library, 3 uL in 20 uL buffer.
• control embryo, restain Friday’s failed sample with L4E24 P2/S2

9:00 am – 7:15 pm

Meetings

• lab meeting (see notes)

embryo staining & imaging

• stained BX-C locus with Bit 1 (9:00 am)
• rinsed out stain
• broke coverslip 1 after it detached from the plastic petri dish before the the nail polish set (2 minutes was not enough)
• started staining slide 2, 2:00 pm
• testing stage
• no staining (at least not near as good a last time with the 15 kb probes).
• stained with bits 1-15 minus 3, (was going to have 3 as a cy3 control). This gives substantial nuclear background and potential faint spots.
• maybe try more stringent wash conditions

Troubleshooting planning

• ordered a cy3 primary (this will help troubleshoot both the probe making and the primary staining).

New stains

• started prep of 2 new coverslips
• ran through complete coverslip prep protocol, including Triton, LN2, Hcl and RNase. now in 50% formamide 2x SCC at 4C.

STORM5

• steve doesn’t work: hal doesn’t save files in the correct directory with the correct name.
• hal doesn’t save position in in the inf file. This makes tracking / returning to position quite difficult. Can use steve to record stage position.

Microscope building

• got quotes from GE for lasers. started new Microscope building project folder
• started excel doc to project costs. should contact Hazen.

9:40 am – 5:00 pm

Goals & progress

• prep and stain embryos with new L7 library. -> DONE
• order food for group meeting. -> DONE.
• pick up suit for interviews. -> DONE.
• work on equipment lists. -> Extracted parts list for STORM5
• get estimates of microscope room specs. -> contacted Mike Paterno
• contact UCSF about dates -> Scheduled: Mar 4th and 5th. (Thur + Fri).
• order Alexa 647 labeled probe complimentary to L7 (thought I did this earlier but check records today and not ordered).

Experiments

• L7 library staining embryos (3 uL library to 25 uL hybe dilution buffer). Stained 2 coverslips.
• denatured with block set to 90C. Block reading 78-80C during incubation once cold glass slides and water are added. Thermometer reading 85C.

Image results

• RNA probes clearly labeled better than smFISH DNA probes for confocal imaging.
  smFISH hb-647

smFISH RNA probes with antibody

Code

• start writing (and streamlining) code for an engrailed locus library.

maintenance stuff

• started new box of common MERFISH supplies for team
• started new excel sheet for logging my lab purchases
• created new excel sheet indexing all parts purchased for STORM5

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.

9:40 am – 7:00 pm

Reading

• Recommendations for junior scientists: https://drmaltman.wordpress.com/2014/09/17/10-simple-steps-to-building-a-reputation-as-a-researcher-in-your-early-career/
  • I don’t have time to make it to the lecture on this today, but some simple suggestions worth bearing in mind from Micha Altman
  • I should probably update my website at somepoint

Goals

• probe synthesis – T7 reactions
  • still waiting for toehold probes to arrive, so there’s no rush on this.
  • also waiting for labeled version of RT primers to arrive, though I could do a test with the unlabeled version.
• embryo smFISH optimization, try Golding protocol.

Thoughts for the day

• can I adapt kindle fire as a cheap, web enabled, bench-top lab notebook device?
• wrote to find out if I have any money left in my fellowship supply fund to test this.

Experiments

Embryo smFISH

• continuing probe making
• precipitate snail-dig probe, dry, resuspend in hybe buffer
• running traditional RNA FISH stain (from xyelenes forward).
• 5 nmol in 100 uL, target is 100 nM total probes (2 nM per probe)
  • so 1 in 500 uL. Try at double, 2 in 500 uL.
• tried and true Levine lab method: using new dig-RNA. 4 uL RNA in 200 uL. Tested on both embryo samples.
  • incubating in shaker at 55C O/N
• Xu method, didn’t have tRNA, ribonucleoside vanadyl complex, or RNase free BSA
  • used instead ssDNA and heparin, with 10% dextran sulfate
  • incubating at 30 C O/N (no shaker available, not as necessary with dextran sulfate, embryos don’t settle quickly).

Probe making

• set up T7 reactions for 2x sample of L7 library and for all probes 1, 2, and 4 with Hazen (+ a positive control)
• sample order: 1, 2, 4, gap, L7, L7, positive control.

10:10 am

(back from UCSD interview)

Probe synthesis

• L7 10kb BXC library — amplifying
  • amplifies and saturates about cycle 24 (ran 27 cycles which was probably extra but should still make good probe)
• purified amplification, ready for T7 reaction
• don’t have new labeled RT primer in yet, need to wait to run RT reaction 🙁