10:30 A
General stuff
- reply to Olivia about quantifying nuclear concentration, mRNA FISH
- Need to fedex keys and forms back to U Chicago. Wrote to Matt to get acct info
- # Enter all accepted Banff Invites into BIRS system, including co-organizers
Probe making round 3
- 1 mL scale PCR of all probes (compare library strength)
-
Try 2x dilution and 1 more round of PCR to try to remove cross annealed probes
-
-
Try 1 uL of ‘pure stuff’ for Black and Green libraries
- Actually works this time, Green a bit smeary.
- Lesson: should pool PCR tubes before gel — there is definetely variation tube to tube (which is nice to diagnose), however for later comparisons we want to see the mixed pool.
| Pos | Sample | Band Size | QuBit | Total DNA |
|---|---|---|---|---|
| A | Blue | smear | 38.5 ng/uL ? | 38 ug in 1 mL |
| B | Yellow | 120,170,220,smear | 51 ng/uL | 51 ug in 1 mL |
| C | Black | no band | 7 ng/uL ?! | 7 ug in 1 mL |
| D | Green | no band | 10 ng/uL ?! | 10 ug in 1 mL |
| E | Ubx | 200 | 10 ng/uL (2) | 5 ug in .5 mL |
| F | AbdA | 200 | 13 ng/uL (2) | 6 ug in .5 mL |
| G | AbdB | 150,200 | 13 ng/uL (2) | 6 ug in .5 mL |
| 1 | Black2 | 110, 150 | 22 ng/uL | 22 ug im 1 mL |
| 2 | Green2 | 150 200 | 32 ng/uL | 8 ug in .25 mL |
Cell culture
- passage Kc167 cells
- new media for Kc167 cells
Embryo in situs!
- 5 min thaw in 2x SSCT with Hoechst
- 7 min quench with .1% sodium borohydride (probably quenches Hoechst?)
- 5 min incubation in 2x SSCT
- Thaw frozen, fresh DI formamdide. Make new 50% formamide 2x SSCT wash buffer.
- 35 min incubation at 60C in prehybe
- 8 uL en probe in 40 uL dilution buffer, used 30 uL on coverglass
- incubating at 37C O/N (Hao also using incubator at 37C).