Cold, Infected Pool with RNeasy and DecaPod S1E13

Today I edited and published S1E13 of DecaPod. In this episode I interview Madi Shipley (MS, SAFS) who works with Bering Sea Tanner crabs. I also extracted RNA from 8 samples (all Cold, infected, Day 26) using Qiagen RNeasy and definitely have enough RNA for a pool!

DecaPod

S1E13 posted

RNeasy Extraction: 8 samples D26, cold, infected

Today I extracted RNA from 8 samples (all the second out of the three that were taken per crab):
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I used the RNeasy protocol, with a few modifications:

  • At steps 5-7, I centrifuged at 10,000 rcf
  • Added 2-ME to Buffer ATL (since 8 samples, did 3mL buffer, 30µL 2-ME)
  • Did not add RNA carrier
  • Eluted with 14µL water (provided in Kit)

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Qubit results look pretty amazing! My bioanalyzer results from last time looked pretty good, so I think we can trust these results. EXCEPT for tube number 413-2 because some of the working solution did not make it into the qubit tube.

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The tubes (now with 13µL of material) are in Rack 5, Column 4, Row 3 in the -80.

NWGC wants 20ng/µL of RNA in 50µL sample (GitHub issue #184). It looks like we have more than enough!

When I return from the short holiday break this weekend, I’ll start picking out samples for the other pools and get feedback from Steven and Sam.

Written on November 21, 2018