10:30 A – 8:35 P
Probe making
- resuspend ethanol precipitated probe samples
- make and cast new UREA gels. 2x 15 well .75mm, 1x 5 well 1.5 mm (25 mL is not enough for 2x 1.5mm — these take 10mL each. the .75 mm take 5mL).
- Running ethanol-precipitated DNA pre-digest vs. ethanol-precipitated DNA post digest diluted samples on .75mm UREA PAGE gel 1 hr. Hopefully the DNA runs into the lanes and looks different this time.
- lane order: ladder, ladder, undigested B,Y,K,U, digested B,Y,K,U, ladder.
Cell staining probe tests
- Polylysine coat 18 mm coverglass in 12 well chambers.
- plate with Kc167 cells. Passage culture cells into new culture flask
- fix cells and permiablize according the 3D FISH protocol.
| probe | primary | secondary |
|---|---|---|
| AATAT P1 | 1 uL of 50 pmol/uL dilution | 1.7 uL S1 |
| No primary | blank | 1.7 uL S1 |
| Blue probe | 5 uL = 50 pmol in 25 uL | 1.7 uL S1 |
| yellow probe | 5 uL = 50 pmol in 25 uL | 1.7 uL S1 |
| black probe | 5 uL = 50 pmol in 25 uL | 1.7 uL S1 |
| green probe | 5 uL = 5 pmol in 25 uL | 1.7 uL S1 |
| ubx probe | 5 uL = 50 pmol in 25 uL | 1.7 uL S1 |
| RNA not complement | 5 uL in 25 uL | 1.7 uL S1 |
| RNA complement | 5 uL in 25 uL | 1.7 uL S1 |
- denatured with reprogrammed heat block set to 85C (still reading a bit low?)
- incubated O/N at 47C
Project 2
- polylysine coat 3 6-cell flow chambers
- Alec plates these with A549 cells.
- allow cells to grow over weekend