RNA isolation protocol and plan for tomorrow

Tommorrow I will be trying out RNA isolation on 2 extra Tanner crab hemolymph samples. Try protocol with 2 samples: 465 and 468 Both samples are yellow (from third sampling day) and are from crabs that are infected, immature, and held at ambient temperatures throughout the experiment.

Protocol is from RNA isolation protocol

Gathering materials/preparing reagents

(1) Prepare 75% Ethanol made with 0.1% DEPC-treated H20 Total volume needed for 2 samples: 800µl —> 1000µl

We have 200 proof ethanol (100%), so will combine: 750 µl ethanol 250 µl DEPC-treated H20

(2) grab: Reagents:

  • RNAzol —> will need 2 ml total for 2 samples
  • 2-propanol —> will need __?__ total for 2 samples
  • 75% ethanol, etc. made above
  • 0.1% DEPC-treated H20
  • Ice —> wet ice ok for short-term storage

PPE: Gloves, safety glasses, lab coat, fume hood

Equipment:

  • Pipette (P1000)
  • Pipette tips (for P1000)
  • Sterile snap cap tubes (2)
  • Microfuge
  • Vortex

PROCEDURE

  1. Add 1ml of RNAzol RT to sample tube
  2. pipette to mix
  3. Vortex 15s.
  4. Add 400uL of 0.1% DEPC-treated H2O.
  5. Vortex 15s.
  6. Incubate at room temperature (RT) for 15mins.
  7. Centrifuge 12,000g for 15mins @ RT.
  8. Transfer 750uL of supernatant (do not disturb pellet) to sterile 1.7mL snap-cap tube. (Discard remaining liquid in RNAzol RT Hazardous Waste container in fume hood. Leave old tube open in fume hood over night and then discard in regular trash.)
  9. Add 1 volume of isopropanol. This means that you add the volume of isopropanol that is equivalent to total sample volume.
  10. Vortex 5s.
  11. Incubate @ RT for 15mins.
  12. Centrifuge 12,000g for 10mins @ RT.
  13. Discard supernatant; do not disturb pellet.
  14. Add 400uL of 75% ethanol, centrifuge 4,000g for 3mins @ RT.
  15. Repeat steps 13 and 14 one time.
  16. Repeat step 13.
  17. Centrifuge 4,000g for 1min @ RT.
  18. Removal residual ethanol.
  19. Immediately resuspend pellet in appropriate volume of 0.1% DEPC-H2O (volume is dependent upon pellet size, but 50uL is usually sufficient).
  20. Keep sample on ice for short-term storage (i.e. no more than 2hrs) or store @ -80C.
Written on February 13, 2018