Sam's Notebook

RNA Isolation - Ronit’s C.gigas diploid/triploid dessication/heat shock ctenidia tissues

by ["kubu4"] ~1 min read

Isolated RNA from a subset of Ronit’s Crassostrea gigas ctenidia samples (see Ronit’s notebook for experiment deets):

  • D01 C

  • D02 C

  • D19 C

  • D20 C

  • T01 C

  • T02 C

  • T19 C

  • T20 C

RNA was isolated using RNAzol RT (Molecular Research Center) in the following way:

  • Unweighed, frozen tissue transferred to 500uL of RNAzol RT and immediately homogenized with disposable pestle.

  • Added additional 500uL of RNAzol RT and vortexed to mix.

  • Added 400uL of 0.1% DEPC-treated H2O, vortexed and incubated 15mins at RT.

  • Centrifuged 12,000g for 15mins at RT.

  • Transferred 750uL of supernatant to clean tube (discarded remainder), added 1 volume (750uL) of isopropanol, vortexed, and incubated at RT for 10mins.

  • Centrifuged 12,000g for 10mins at RT.

  • Discarded supernatant.

  • Washed pellet with 75% ethanol (made with 0.1% DEPC-treated H2O).

  • Centrifuged 4,000g for 2mins at RT.

  • Discarded supernatant and repeated wash steps.

Pellet was resuspended in 50uL of 0.1% DEPC-treated H2O and stored @ -80oC in Ronit’s temporary box.