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. (Put in -20C on labeled tube rack in FTR 209)
  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

qubit_tube_conc_ng.ml original_sample_conc_ng.ul sample_vol_ul dilution_factor tube_number extraction_method ul_sample-used elution_vol_ul total-yield_ng
39.5 3.95 2 100 41 Zymo_microprep 35 15 51.35
126 12.6 2 100 50 Zymo_microprep 35 15 163.8
200 20 2 100 111 Zymo_microprep 35 15 260
136 13.6 2 100 108 Zymo_microprep 35 15 176.8
62.3 6.23 2 100 121 Zymo_microprep 35 15 80.99
78.4 7.84 2 100 34 Zymo_microprep 35 15 101.92
31 3.1 2 100 48 Zymo_microprep 35 15 40.3
155 15.5 2 100 53 Zymo_microprep 35 15 201.5
Out of range Out of range 2 100 32 Zymo_microprep 35 15 #VALUE!
249 24.9 2 100 119 Zymo_microprep 35 15 323.7
293 29.3 2 100 10 Zymo_microprep 35 15 380.9
148 14.8 2 100 91 Zymo_microprep 35 15 192.4
54.5 5.45 2 100 19 Zymo_microprep 35 15 70.85
160 16 2 100 8 Zymo_microprep 35 15 208
Out of range Out of range 2 100 62 Zymo_microprep 35 15 #VALUE!
160 16 2 100 103 Zymo_microprep 35 15 208
37.6 3.76 2 100 109 Zymo_microprep 35 15 48.88
163 16.3 2 100 163 Zymo_microprep 35 15 211.9
Out of range Out of range 2 100 27 Zymo_microprep 35 15 #VALUE!
41.8 4.18 2 100 101 Zymo_microprep 35 15 54.34
310 31 2 100 81 Zymo_microprep 35 15 403
143 14.3 2 100 144 Zymo_microprep 35 15 185.9
235 23.5 2 100 133 Zymo_microprep 35 15 305.5
Out of range Out of range 2 100 15 Zymo_microprep 35 15 #VALUE!

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

(Put remaining hemolymph pellet tubes in Rack 8, column 3, row 5 in -80 (the nearly empty box from first day sampling tubes)).

Written on October 25, 2019