Kaitlyn’s notebook: C. baridi hemocyte pellet RNA isolation and quantification

Samples

Isolated RNA from the following hemolymph pellet samples:

  • 58
  • 61
  • 64
  • 72
  • 79
  • 93
  • 94
  • 96
  • 99
  • 110
  • 114
  • 126
  • 140
  • 155
  • 165
  • 173
  • 180
  • 208
  • 252
  • 256

Samples 114 and 126 were previously done successfully, but due to changes in spreadsheet organization, they were repeated accidentally.

The following samples could not be found, all of which have green caps:

  • 6
  • 43
  • 76
  • 130

Samples 58 and 61 were clear (left) in contrast to the cloudy appearance of the other samples (right). 20200127_134701.jpg

RNA Isolations

Isolated RNA using the Quick DNA/RNA Microprep Kit (ZymoResearch) according to the manufacturer’s protocol for liquids/cells in RNAlater.

  • Used 35uL from each RNAlater/hemocyte slurry.
    • Except for sample 58 where all 20ul of sample were used
  • Mixed with equal volume of H2O and 8x lysis buffer
  • Retained DNA on the Zymo-Spin IC-XM columns for isolation after RNA isolation.
    • Held at 4C
  • Performed on-column DNase step.
  • RNA was eluted in 15uL H2O

RNA quantification: HS Assay on Qubit

RNA was quantified on the Roberts Lab Qubit 3.0 using the RNA High Sensitivity Assay (Invitrogen), using 2uL of each sample.

    • For samples 58 and 61 which registered LOW:
      • Added 2ul more for for a total of 4ul to assay tube
        • still too low

Samples are currently in box in front of Rack 9 (all racks in -80C are full), but I will add to the Shellfish RNA boxes!