Wednesday 05/23/15

9:00 am – 5:30 pm, 9:00 pm – 10:00 pm

Morning tasks

Finish ultra-cryo cleanup (put knives away)
Discuss drive issue with Dell.
- system currently configured with only 4 bays active
- need RAID controller to connect 8 bays together and pass those to the motherboard
- this requires reformatting the drives.

move from methanol to ethanol (through 50:50)
move to xylenes (through 50:50)
treat in xylenes, 1.5 hours
move to ethanol
move to PBS
post fix, 25 min in 5% PFA in 1/2 PBT
8 min Sodium borohydride treatment
rinse in PBT
Trying Little’s pre-hybe buffer: 4x SSC, 35% formamide, 0.1% Tween-20, 10 min
- in 100 mL: 20 mL 20x SSC, 35 mL formamide, 1 mL 10% Tween-20. rest H2O
- in 50 mL: 10 mL 20x SSC, 17.5 mL formamide, 0.5 mL 10% Tween-20, fill to 50 mL
Trying Little’s hybe buffer: 4x SSC, 35% formamide, 10% dextran sulfate 2ug/mL BSA, 0.1 mg/mL salmon sperm, 2 mM ribonucleoside vanadyl complex, 0.1% Tween-20 + 1 nM per probe.
- in 10 mL: 2 mL 20x SSC, 3.5 mL formamide,2 mL dextran sulfate, 100 uL 10mg/mL salmon sperm, 100 uL 10% Tween-20.
probes ship at 5 nmols
that’s ~50 probes, so dissolving this in 100 uL each probe is at 1 uM.
so let’s dissolve in 200 uL RNase / DNase free water, then dilute at 1:500 for experiments. — actually, this stock looks much more dilute than I expect, let’s try 2:500 (not that this will make a difference relative to 1:500).