8:50a – 4:40p
STORM
- Finish O/N STORM of G4
- start splitdax
Probe making
- RNA cleanup using Beckman RNAClean XP beads. Samples F1-F5, F7-F9. Tube format
- F1 yield ~1,500 ng/uL in 25 uL (37.5 ug of RNA). F2 yield ~700 ng/uL (18 ug). Comporable to column yields in 100 uL
- most samples essentially no yield — 10 – 30 ng/uL (might be DNA template). Not sure what happened / why these failed.
staining
- untested probes: F1, F2, F6. Possibly E6 and E7?
All small regions ( < 20kb)
- E02 13 kb green (between R/Y)
- E03 BLUE 13 KB (no genes, good PC/Psc)
- E04 BLUE 10 Kb (flanked by strong PolII)
- E06 Epc (12 kb YELLOW)
- E07 Tou (15 kb YELLOW)
- E09 RED 8 kb intronic tRNA locus, in between blue
- F02 BXC_Y_left (15 kb)
- F08 RED 7 kb region next to black 1, tRNA locus
- F09 BLACK 18 kb region next to RED1
- F12 Taf1_(17kb yellow/green)
- G01 LabRegion_(Lab_Zen2) [83 kb but missing piece of ANT-C]
- G07 AlphaTub84 (5 kb yellow)
-
D12 YELLOW 385kb region [Missing on plate reaction for some reason]
-
orthogonal primers / negative control
PCR
- Add master-mix last because of 3-.5′ exonuclease activity will degrade ssDNA (primers and library).
- 10 uL of 5 uM common primer
- .5 uL 200 uM T7-index primer
- 1 uL of 1:20 library
- 38 uL ddH2O
- 50 uL of Phusion master mix.
- 32 cycles instead of 29.
- seems to have finished very fast, and neighboring block is doing something weird (step 98C, but set at -100C forever)
- check on gel and maybe run again.
Cell culture
- new media for 75cm^2 flasks of Kc167 cells.