Today I finished speed vac-ing (medium heat) the pooled sample. It ended up being too low of volume (14.1ul), so I added 40.9ul of 0.1% DEPC-treated H20. I ran 2ul of the sample on Qubit (RNA HS), and got a reading of 20.4 ul (1,081 ng RNA in the sample)!!!! We FINALLY have a sample to send off for library prep and sequencing! After getting info from NWGC (Chris in the Nickerson Lab at Foege) I put the sample on dry ice and walked it over! It is now in their hands until we get the data back. :)
Sample prep
Yesterday wasn’t enough time on the speed vac. So I put it back on (on medium) at 10:10 am. I took it off at 1pm.
I checked the volume of the sample by sucking it all up in a pipet tip (set to 50ul). Then, I decreased the volume on the pipet tip until the liquid was at the tip. It showed that the volume was 14.1 ul. It needs to be at least 50ul. So I sucked the sample back up with the pipet set at 14.1 ul. It all fit.
Then I added 40.9ul of 0.1% DEPC-treated H20 so the final volume was 55ul and vortexed it for 10s.
I then used 2ul of the sample to run on the Qubit (RNA HS) to check the RNA quantity. It read as having 20.4ng/ul of RNA! Meaning that in the sample, we had ~ 1,081 ng of RNA!!! (NWGC requires a minimum of 1000ng of RNA in a sample at least 50ul in volume).
I contacted NWGC to get info on what all they needed before I walked the sample over.
I brought it over on dry ice and handed it to Chris Frazar in the Nickerson Lab in Foege at 2:45pm.
He emailed me and Steven (I cc’ed Sam) to get more info on the sample and what kind of sequencing we want. More information on that to come. They will do the library prep and the sequencing.
I am beyond excited we finally have a sample at NWGC!!
Next steps
- Try Tri-reagent method of RNA isolation once the lyophilizer is fixed
- Perform qPCR assay to test shellfish primers (GitHub Issue #353)