10:00a – 8:30p, 10:00p – 12:00a
- finish abstract for Alvaro
- work on slides for Stowers
- STORM with Bogdan finish F1, start coverslip 4
- STORM data processing with Bogdan
- Oligo Prep G3-G5 P1A405, P2Cy3, P4Cy3 samples
- Hybridizations for next small regions
- copying F1 data to ProRAID
Analysis of yesterday’s results
- ~15 ug RNA at 100 bp ~ 450 pmol.
- 500 pmol primer
- 10 mM dNTP = 10 nmol /uL. 6uL = 60,000 pmol. At ~25 of each base per probe that enough for 2,400 pmol
New T7 reactions
- 4 samples at .25x buffer 1x Enzyme. 1-4 on strip. G1, G2, G9, F11. Concentrations: 53, 70, 45, 40 ng/uL
- 4 samples at .75x buffer .75x Enzyme: 5-8 on strip. F8, F7, E4, E3. Concentrations: 70, 47, 60 and 30 ng/uL
- G3-G5 P1A405, P2Cy3, P4Cy3 samples
- forgot to empty dry spin, added elution water and eluted into sample
- G4P4 had too much volume, clearly wash-buffer contamination. Added more binding buffer and ethanol and reran.
- other samples eluted in 15 uL and had ~14 uL volume in resevoir, so hopefully not much contaminated. Used these as probe.
- G3-P1-405 (3 uL) + S1-Cy5 (.7 uL) and G4-P2-cy3 (3 uL) + S2-Cy5 (.7 uL)
- G3-P2-cy3 (3 uL) + S2-Cy5 (.7 uL) and G4-P1-405 (3 uL) + S1-Cy5 (.7 uL)
- G3-P2-cy3 (3 uL) + S2-Cy5 (.7 uL) and F9-P1-405 (3 uL) + S1-Cy5 (.7 uL)
- Discussion with XZ (see private notes)
- Team / co-advising meeting with XZ
- reorganized all freezer stocks. Consolidated all samples into upstairs freezer, downstairs freezer by confocal and downstairs freezer by -80C
- Combined stock aliquots into common box: IPTG, X-Gal, Salmon Sperm, Heparin, Glycerol, STOP Soln, Carb Soln
- Moved all embryos to deep storage (-20C next to -80C).
- more T7 should come tomorrow
- ordered more large scale supply of Phusion
- ordered more Oligo Prep buffer and DNA Wash buffer
- need to validate cleaning protocol for CC-5 columns