9:45A – 10:00 P
Lab meeting (notes)
- scored 5 more back-cross lines (no rescues still):
- [spreadsheet 0AjSqkxgziU1YdENuMzFjejM0TnNXUEQ1OWdHRW1OY1E 602 202 sheet = 1]
- Flipped and watered bottle cross of Df/CyO,esc x Sp/esc males.
- New bottle cross of Df/CyO,esc x Sp/esc males
- Test crosses to shRNAs
- MTD x Psc 2
- MTD x Pc 2
- MTD x SuZ 12
- (MTD virgins somewhat old but plentiful)
- MAT x Psc 2
- MAT x Pc 1
- MAT x SuZ 12
- F1 Psc 2 x MTD at 29C
- F1 Rad21 x MTD at 29C
- F1 Pc 2 x MTD at 29C
- cleared bottles for virgin collection:
- Moved BSC flies in with stocks (need a new flat to fit all stocks in one).
- rewrote STORMrender multicolor load to cope with new image format, use new global drift correction and new version of chromewarp.
- Rewrote Im3D code (called by the Render3D function of STORMrender) to cope with multichannel. Multichannel inputs are listed as cell arrays.
- updates to BXC + K27me3 (see post)
Probe making: measure probe concentrations
- measuring absorbance on Nanodrop UV-Vis
- 500 kbp probe: 650 = 0.50. 260 = 1.388
- En probe 1: 650 = 0.691, 260 = 1.835
- AATAT aliquot 650 = 1.959, 260 = 2.142
- Computing concentrations
- (1.388) 50 mg/mL *1000/(.66*32) = 2.05 pmol/uL
- Passage all cell culture stocks (KC167, S2, S2R+)
- 5 coverslips (22x22mm) coated with polylysine (new stock, brief coat and remove), in a petri dish of diluted S2 cells — 6:15 P. Allowing to adhere prior to fixing for in situ.
- Start warming coplin jars: drying oven set to 92 C. Small incubator set to 42C.
- Added 12 uL of En probe, 12 uL of 500 kbp probe, and 5 uL of AATAT probe to slide. placed coverslip on top and sealed with rubber cement (all at RT).
- Meanwhile heating up a heat block + water bath (styrofome box) to 92C inside large hybe oven. Heat block and waterbath definitely do not reach 92C. Need to do this in an actually heated waterbath.
- incubate at 42 while heating up heatblock to 92 for traditional ‘on the heat’ version of denaturing (1 min). Will try to follow up with Alec about getting a proper small water bath that can run to 92C.