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 |
- Let pelleted hemolymph thaw, then vortexed for a second.
- 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).
- Added 35ul of RNase free water
- Added 280 ul Lysis buffer
- Mixed all 350ul of liquid by vortexing.
Purification
- Transfered 350ul of sample into yellow spin column.
- Spun 10,000 g 30s.
- Saved DNA on the column for later.
- Saved flowthrough with RNA and continued
- Added 350ul 100% ethanol and pipet to mix.
- Transfer all 700ul to clear spin column.
- 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.
- 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
- 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.
- Added 400 ul DNA/RNA wash buffer. Spin 10,000 g 2 minutes. Transfered column to fully labeled RNase-free snap caps.
- 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.
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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