This post contains my notes on the Zymo Research Protocol for Quick-DNA/RNA Microprep kit, and my extraction plan for this next week as I try to get enough RNA to get two more libraries (Day 9 infected (1), and Day 9 uninfected (2)).
Sample extraction plan:
Remaining Day 9 sample tubes (reminder: no temperature treatments were happening on Day 9):
- 39 uninfected (21 are listed as “M” for “mature”)
- 49 infected (all are “I” for “immature”)
I will start out by extracting 12 infected and 12 uninfected samples on my first extraction day (tomorrow or Thursday). I will use the Qubit to keep track of the yields.
The next day I’ll do another 12 infected and 12 uninfected samples (Thursday or Friday). If there are still not high enough RNA yields, I’ll do another round of extractions of 12 infected and 12 uninfected (Friday).
Hopefully by next week I’ll have the ability to create two pooled libraries for Day 9: (1) infected Day 9, and (2) uninfected Day 9.
My samples are crab hemolymph (and Hematodinium spp.) cells. The samples were originally stored in RNAlater, then in Winter 2018, I spun them down, and saved the supernatant in other sample tubes.
- Add 96 ml 100% ethanol (or 104ml 95% ethanol) to the 24 ml DNA/RNA Wash Buffer concentrate
- Add 275 ul DNase/RNas-Free Water per vial to reconstitute the lyophilized DNase I at 1 U/ul. Mix by gentle inversion. Store frozen aliquots at -20C.
- Add 1040 ul Proteinase K Storage Buffer per vial to reconstitute the lyophilized Proteinase K at 20 mg/ml. Vortex to dissolve. Store at -20C.
Cells are already “pelleted”.
Resuspend cell pellet in DNA/RNA Lysis Buffer (400 ul).
- Transfer sample to a Zymo-Spin IC-XM Column in a Collection Tube and centrifuge (10,000 g for 30 sec)
- Add an equal volume (400 ul) of ethanol (95% or 100%) to the flow through and mix well. Transfer to a Zymo-Spin IC Column in a Collection Tube and centrigure (10,000 g for 30 sec). Discard flowthrough.
- Add 400 ul DNA/RNA Prep Buffer to the column and centrifuge (10,000 g 30 sec). Discard the flowthrough.
- Add 700 ul DNA/RNA Wash Buffer and centrifuge (10,000 g 30 sec). Discard flowthrough.
- Add 400 ul DNA/RNA Wash Buffer and centrifuge 10,000 g for 2 minutes to ensure the removal of the wash buffer. Carefully transfer column into a clean microcentrifuge tube.
- Add 15 ul DNase/RNase-Free Water directly to the column matrix. Let stand for 5 minutes, then centrifuge (10,000 g 30 sec) to elute RNA from column.
Use 1 ul on the Qubit to quantify RNA.
Place remainder of sample into -80 C.
Save Qubit results, input sample tube numbers, add date to master-qubit.xlsx, then
join sample_table.csv using this R script (081919-sample-qubit-master.Rmd). This will update the master qubit sample spreadsheet: master-qubit-sample.csv.