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.