Today I extracted RNA from 10 samples from Day 26. These still had all three samples in the box, so they are all ###-1 tubes. The QUbit wouldn’t let me download the data from the USB. I’m not sure why, but luckily I wrote down, as I always do, the reads for each tube number. Out of the 10, 7 had quantifiable RNA by the RNA HS Qubit.
Samples used:
Tri-reagent extraction protocol
- Added 1mL TriReagent to each tube
- vortexed to mix/dissolve solute
- incubated 5mins at RT
- added 200uL of chloroform
- vortexed 15s to mix
- incubated at RT for 5mins
- centrifuged 15mins, 12,000g, 4oC
- transferred aqueous phase to new tube
- added 500uL isopropanol to aqueous phase
- mixed, incubated at RT for 10mins
- centrifuged 8mins, 12,000g, at RT
- discarded supernatant
- added 1mL 75% ethanol
- centrifuged 5mins, 12,000g at RT
- discarded supernatant
- resuspended in 10uL of 0.1% DEPC-treated H2O.
Thoughts from today’s extraction:
- It was more difficult to see any RNA at the bottom of the tubes in the end. Friday was a lot easier.
- One of the 10 tubes did not have a clear aqueous phase separation. I had to remove some of the pink off the top, then attempt to just get the clear liquid beneath that and above the rest of the pink. I did not write down which tube it was, so I’m not sure which Qubit result belongs to it.
Difficulty getting Qubit data off of the USB
I re-downloaded the data on the Qubit a second time, but still I couldn’t download it from the USB. I kept getting the same error, and I would retry multiple times, but still wasn’t able to get it. I do need that data so that I can add it to my sampling master spreadsheet!
Need to do bioanalyzer on these 10 samples
I ran out of time today - I have to go pack and get some other things done before I leave tomorrow. May ask someone else to bioanalyze, or I’ll do it myself when I return next week.
The samples are in purple tubes in Rack 5, Column 4, Row 3:
Qubit results from today (got off the USB 10/19/18)
here