Today Steven, Sam, and I met to talk about the issue with poor results on the QUbit and Nanodrop1000 on the pooled hemolymph samples I prepared. Short-term plan is to try out the RNA protocol, but multiply all reagents times 6 (so, use 6ml of RNAzol, etc.) to see if that ratio would help; run the Bioanalyzer on the samples that Sam processed as well as some random samples that I isolated. Today, I did the 6x RNA protocol on 4 of the extra samples from day 26 (samples from day 26 were taken in triplicate).
RNA Isolation Protocol x 6
Link to the protocol I did today: HERE
*Note: I forgot to do step 6
It was a bit challenging today because in order to support those larger volumes of reagents, I had to use 13ml round-bottomed tubes. It was difficult to see any pellets or genetic material, but I did my best. I oriented the tubes in the same way every time I put them in the centrifuge and I marked the orienation on the tubes so that I knew where the genetic material should be if it was going well.
Per Sam’s suggestion, I resuspended the samples in 25ul of 01% DEPC-treated H20, instead of the typical 50ul. I also pipetted to mix/dissolve.
I ran the Qubit, and they were ALL “out of range/too low”.
I did not run the Nanodrop, because I can’t remember how to use it. I will do it first thing tomorrow when Sam is in.
The tubes I processed were:
- 411 –> COLD, INFECTED (note that it died before RNA sampling, suspect 219 tube OK, and has 3182/3184, but no way to be sure (I don’t understand this note - can ask Pam to clarify))
- 423 –> COLD, UNINFECTED
- 503 –> AMBIENT, INFECTED
- 515 –> AMBIENT, UNINFECTED
Goals for tomorrow:
- Run Nanodrop on the samples I isolated yesterday
- Run Bioanalyzer on the samples Sam isolated RNA from as well as some random ones selected from what I’ve already done
- Try out/read about using Tri-reagent for isoalting RNA