9:45a – 5:15p, 10:00p-12:25p
General stuff
- mailroom still closed (10a) still can’t pick up package
- need to get PCR tubes, razor blades and 10x PBS from stock room.
cell staining
- hot wash 65C in waterbath 17 min.
- no staining (low background, just isolated dyes scattered throughout, a few maybe brighter, but not convincingly)
- 405 background still high.
- should have been 60C, maybe too strong hot-wash? Previously have done air incubator not waterbath. Try to restain with more probe?
Restain
- ubx: 8 uL primary + 6 uL secondary + 0.2 uL tertiary + 10 uL formamide + 2 uL 50% dextran sulfate
- abdA: 10 uL primary + 3 uL secondary + 0.2 uL tertiary + 10 uL formamide + 2 uL 50% dextran sulfate
- (note, effectively secondary diluted to 1.5:20 after volume was raised to 40 by adding more formamide. We are in only 1x SSC)
RT reaction
- ubx 20, abdA 20, abdB 20, no-primer, ubx-primer + RNA no RT
- 300 pmol primer (3uL)
oligo cleanup
- 3 RNaseA digests + 3 controls
- 3 new RT reactions + 2 controls (after alkaline hydrolysis).
concentrations
- ubx 20ug new = 792
- abdA 20ug new = 557
- abdB 20ug new = 240
- no Enzyme = 272 (300 pmol primer)
- no primer = 128 (non degraded mRNA)
- ubx RNase = 185. ubx = 208 no RNase
- abdA RNase = 168. abdA = 196 no RNase
- abdB RNase = 75. abdB = 97 no RNase
add RNase A (10ug/mL diluted 1:20 and then 1:20 15 in elution) to U20, A20, B20, no Enzyme and no Primer. Let react O/N at RT,