Grace’s Notebook: Crab RNA extractions with Qubit results

Today I ran 2ul of the 24 samples I extracted yesterday. 20 out of 24 had RNA. Details in post.

Prep

Labeled tubes

  • 24 RNase-free snap cap tubes (for preparing the 35ul sample)
  • 24 RNase-free snap cap tubes (with C. bairdi RNA, tube number, and date)
  • 24 yellow zymo spin column lids (Next time also label collection tube)
  • 24 clear zymo spin column lids

Grabbed 100% ethanol in a 15ml tube, and RNase-free water in a 15ml tube.

P1000, P200, P20 and tips

Sample prep

Tubes I grabbed:

status tube number
infected 15
infected 133
infected 144
infected 81
infected 101
infected 27
infected 163
infected 109
infected 103
infected 62
infected 8
infected 19
uninfected 91
uninfected 10
uninfected 119
uninfected 32
uninfected 53
uninfected 48
uninfected 34
uninfected 121
uninfected 108
uninfected 111
uninfected 50
uninfected 41
  1. Let pelleted hemolymph thaw, then vortexed for a second.
  2. Transfered 35ul of the slurry to a labeled snap cap tube (some tubes (27, 62, 19) only contained ~35ul, whereas other tubes still had a lot left).
  3. Added 35ul of RNase free water
  4. Added 280 ul Lysis buffer
  5. Mixed all 350ul of liquid by vortexing.

Purification

  1. Transfered 350ul of sample into yellow spin column.
  2. Spun 10,000 g 30s.
  3. Saved DNA on the column for later.
  4. Saved flowthrough with RNA and continued
  5. Added 350ul 100% ethanol and pipet to mix.
  6. Transfer all 700ul to clear spin column.
  7. Spun 10,000 g 30 s. Discard flow-through

At the end of Step 7, there was white crystal-y stuff in tubes 144, 8.

  1. Added 400ul DNA/RNA prep buffer. Spun 10,000g 30 s

At end of Step 8, tubes 15, 10, 53, 121 still had some liquid on the column

  1. Add 700 ul DNA/RNA wash buffer. Spin 10,000 g 30s

At end of step 9, 163, 10, 121, still had liquid on column. Spun these again at 10,000 g 30 s. Then they were fine.

  1. Added 400 ul DNA/RNA wash buffer. Spin 10,000 g 2 minutes. Transfered column to fully labeled RNase-free snap caps.
  2. Added 15ul RNase-free water to column matrix and let sit for 5 minutes. Spun to elute 10,000 g 2 minutes.

Put samples in -80C over night becuase I left.

Qubit

Ran 2ul of each sample on Qubit.

20/24 samples had RNA.

Link to data

Extracted RNA is in -80, Rack 7, col 3, row 4

(Need to move hemolymph pellets back to their respectve boxes still. Currently hemolymph pellets from this extraction are in the -80 in the same box as the pelleted RNA.)

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