10:00 am – 5:15 pm 10:45 pm – 11:30 pm
Tasks
- discussed with Dell again about memory (they make this unnecessarily complicated).
Experimental goals for today
- check progress RNAi knockdowns
- purify more RNA for RNAi
- set up new RNAi knockdown with more controlled
Ordering
- sent Alec quote from Dell
- ordered Qiagen RNeasy kit recommended by SN
- ordered superscript III first-strand synthesis kit
- (might be able to borrow a few reactions from Jeff/Steven)
- Biosearch probes for hb arrived today. Yay! (need to prep some embryos to test).
RNAi experiments
RNA cleanup
remove template DNA
- first, DNase digestion of template following NEB protocol
- “To remove template DNA, add 30 μl nuclease-free water to each 20 μl reaction, followed by 2 μl of DNase I (RNase-free), mix and incubate for 15 minutes at 37°C.”
cleanup with Agincourt (Beckmann-Coulter) RNA-clean XP beads
- online protocol
- Add 1.8 volumes of beads to sample in 1.7 mL tubes (min sample volume 50 uL + 90 beads)
- Mix by pipetting, incubate 5-20 minutes at RT (not on magnet)
- Separate on magnet until clear
- discard solution
- rinse 3x in 70% ethanol (500 uL – 1 mL)
- let dry 10 minutes
- Elute in >30 uL RNase free water
Quality checks
- RNA concentrations all in the ~1000 ng/uL – 1500 ng/uL range according to nanodrop
- running on 5% (2 month expired) TBE Urea PAGE gel
- also forgot to remove tape. Still getting substantial product trapped in wells. Both RNA and DNA ladder look smeary. (somewhat old DNA ladder, maybe it is also a bit degraded). Still, hard to explain the larger than band smears based on degradation when running a denaturing gel.
RNAi knockdown progress
- all wells 100% confluent
- no visible differences between wells yet
Cell culture
- cells in Schneider’s + 10% FBS are not adhering
- resuspend 11 mL of confluent cells in Schneider’s without FBS.
- Add FBS to 4 mL of this mixture (400 uL to 4 mL). Plate cells on 75mm2 flasks and bring volume up to 15 mL complete Schneider’s (with 10% FBS)
- RNA not ready. Resupended all cells in complete media
- cells in complete media still not adhering. Hopefully they grow at least and we can spin-down when necessary.
- plated cells on 18mm coverglass freshly coated with Poly-D lysine, (and UV sterilized) in 12-well plates (~2:45 pm). Low density but workable.
Heat shock experiments
- heat shocked for 20 min at 36.5C the newly plated Kc cells (after about 1 hour to attach).
- fixed cells at 36.5C.
- prepping cells for DNA-FISH. Now in glycerol at 4C