Notes on protocol and plan for Crab Project extractions - use info from Sam's notebook posts

This post contains my notes on the Zymo Research Protocol for Quick-DNA/RNA Microprep kit, and Sam’s notebook post to create a protocol for extracting RNA. I also include 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 (Thursday, 10/24/19). I will use the Qubit to keep track of the yields.

The next day I’ll do another 12 infected and 12 uninfected samples (Friday, 10/25/19). If there are still not high enough RNA yields, I’ll do another round of extractions of 12 infected and 12 uninfected (Friday, 10/25/19).

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.

Protocol plan:

(From Zymo Research Protocol for Quick-DNA/RNA Microprep kit and Sam’s notebook post)
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.

Prepping reagents (Only have to do if I open a new kit)

  1. Add 96 ml 100% ethanol (or 104ml 95% ethanol) to the 24 ml DNA/RNA Wash Buffer concentrate
  2. 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.
  3. 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.

Sample preparation:

Cells are already “pelleted”.

  1. Transfer 35 ul of slurry to a microcentrifuge tube (vortex first).
  2. Add 35 ul of RNase-free water.
  3. Add 140 ul of DNA/RNA Lysis Buffer and mix.
    In total, tube contains 210 ul of liquid

Purification:

  1. Transfer sample to a Zymo-Spin IC-XM Column in a Collection Tube and centrifuge (10,000 g for 30 sec). SAVE FLOW-THROUGH
  2. Add an equal volume (210 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.
  3. Add 400 ul DNA/RNA Prep Buffer to the column and centrifuge (10,000 g 30 sec). Discard the flowthrough.
  4. Add 700 ul DNA/RNA Wash Buffer and centrifuge (10,000 g 30 sec). Discard flowthrough.
  5. 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.
  6. 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.

RNA quantification:

Use 1 ul on the Qubit to quantify RNA.
Place remainder of sample into -80 C.

Spreadsheet:

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.

Written on October 23, 2019