9:50a – 1:00a
- 10a – 2p
- see notes
- cy7 dye from Lumipore arrived
- A750 labeled IDT primers arrived
- first test gel — try old gel of Bogdan’s (11/18). Ran smeary and fast, primers ran off the bottom
- reran with precast gel (forgot to take tape off at start, removed after first hour, so gel ran a bit squished. Not a good day for gels)
- substantial primer bands still visible, except in lane 5. Why are 2 and 3 the strongest? 1 I guess is a bit spread out. Seem dilution helps.
- G5 is still reproducibly creating a double band. I don’t like this.
More RT tests
- samples 3 + 4
- 2 uL primer, 6 uL RNA, 3 uL new dNTPs, 65C denature, ice anneal, 42C incubation. Hot Start.
- 2 uL primer, 6 uL RNA, 3 uL new dNTPs, 65C denature, ice anneal, 50C incubation. Hot Start.
- (4 uL 5x buffer + 1 uL RNasin ) x5 + 1 uL Maxima
- staining failed again! Despite high concentration matches previous successful staining just a few days ago.
- Maybe the hybe solution got too diluted with 2 probes both added in large volumes?
- Try 3 uL hybes, with positive controls with water added instead of P3 / S3.
- F11-P1 (3uL) + .7 uL S1-A647 + 3.7 uL ddH2O.
- F11-P1 (3uL) + Fll-P3 (3uL) + .7 uL S1-A647 + .7 uL S3-A750
- Fll-P3 (3uL) + .7 uL S3-A750
- G1-P1 (3uL) + .7 uL S1-A647 + 3.7 uL ddH2O.
- G1-P1 (3uL) + Gl-P3 (3uL) + .7 uL S1-A647 + .7 uL S3-A750
- Gl-P3 (3uL) + .7 uL S3-A750
- Analyzing D07 data (9kb green transcribed)
- highly variable, pretty messy, some spots have lots of density
- sometimes round symmetric blur, 3K localizations
- E02, 13 kb green domain
- concentrated, punctate, symmetric
- data only goes up to 0_5, missing last 4 cells.
- Running E11 bead fitting (somehow didn’t get analyzed) on Tuck
- Running E03 dot and bead fitting on Tuck (started 11pm)