Bairdi hemolymph extraction plan (work in progress)

Here’s the general extraction plan after feedback from lab meeting today. I am planning on getting started on extractions next Tuesday (I’ll be done with end-of-quarter work and my online SCUBA class by then!). (Edited this post a lot since my original post this morning)

Number of samples to process

Based on the two most recent RNeasy trials (Feb 15th, and Feb 20th), we saw that using 10ul of the slurry yielded more RNA per ul of slurry as compared with using 50ul samples. We don’t know what the best sample volume is.

I’ll use 15ul of the hemolymph slurry and process 12 samples at a time, one from each treatment/infection status
Just start doing all the tubes. Don’t have to wait for the kits to arrive since we still have some. I should make sure that we have the Qubit RNA HS kits are in, though.

The goal is to get to the point where I have 12 pooled samples, each with at least 1000ng RNA in 50ul of DEPC-treated H20.

Protocol preparation

Make solutions fresh before each extraction.

Make:
70% ethanol
80% ethanol
Buffer RLT plus B-ME

Sampling: Thaw the hemolymph sample on wet ice
Vortex to mix slurry up
Sample out 15ul of the slurry for processing

RNeasy

  1. Add 350ul of Buffer RLT Plus + B-ME solution
  2. Vortex to mix
  3. Transfer lysate to QIA Shredder column with 2ml collection tube. Centrifuge 2min at full speed
  4. Transfer flow-through to gDNA Elimninator column with 2ml collection tube. Centrifuge for 30s at 12,00 g. Discard column.
  5. Measure the amount of flow-through for both samples and added that same volume’s worth of 70% ethanol. Mix by pipetting.
  6. Transfer sample (including any precipitate that may have formed) to RNeasy MinElute column (from fridge in 209). Close Lid. Centrifuge 20s at 12,000g. Discard flow-through.
  7. Add 700ul Buffer RW1 to RNeasy column. Close lid. Centrifuge 20s at 12,000g. Discard flow-through.
  8. 500ul Buffer RPE to RNeasy column. Close lid. Centrifuge 20s at 12,000g. Discard flow-through.
  9. Add 500ul 80% ethanol to RNeasy column. Close lid. Centrifuge 2min at 12,000g. Dsicard collection tube and flow-through.
  10. Put RNeasy column in new 2ml collection tube. Cut off RNeasy column lid. Keep tube open and centrifuge at full speed for 5min. Discard flow-through.
  11. Put RNeasy column in new 1.5 ml collection tube. Add 14ul RNase-free water to center of membrane. Close lid. Centrifuged for 1min at full speed.

Qubit
Run 1ul of each sample tube on the Qubit using RNA High Sensitivity
KEEP TRACK OF THE TUBE NUMBERS

Upload the Qubit results from each set of 12 immediately to my laptop, add the tube numbers manually, and save.

Things to do

Current thoughts are to get this started next Tuesday.

Before that happens, I need to:

  • Do calculations for the ethanols and Buffer RLT plus B-ME solutions that need to be made
  • Organize samples that I’ll be extracting from (12 at a time, one from each treatment and infection status)
  • Label boxes
  • Clean up and clear up the protocol- print it out for whoever is around to help me
    • Think about what tasks are assignable to people if anyone is able to help
  • Make sure I have supplies and space to get started
  • Aliquot out DEPC-treated H20??
Written on March 14, 2019