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Total RNA Isolation from S. cerevisiae

Laboratory protocol

The cells were collected and the pellet were resuspended in 3.6 ml AE Buffer (AE=50mM NaAcetate pH 5.2, 10mM EDTA) and then transferred in 15 ml tubes wit 200 µl 10% SDS (w/v). 2 ml of preheated acid phenol (pH 4.3) were added and the falcon were mixed by vortex. After 10’ of incubation in 65 °C water bath, the samples were incubated on ice and then centrifugated at 5000 rpm in an tabletop centrifuge.
The supernatants were transferred to a new tube and added 2 ml of acid phenol to repeat the extraction. After the centrifugation (10’ at 5000 rpm) the supernatants were transferred into a pre-spun (5’ at 1500 rpm) 50 ml Phase Lock Gel tube (Eppendorf 0032 005.330) and 4 ml of chloroform (Fischer BP1145-1) were added into the falcon. After the spin (10’ at 3,000 rpm), the supernatants were precipitated in RNase-free centrifuge tube by adding 1/10 volume 3 M NaAcetate (pH 5.3) and 2.5 volumes of cold ETOH 100% overnight at -20°C. Then, after a centrifugation (30’ at 12000 rpm) the pellets were washed with 70% Ethanol two times. When the pellets were dry, they were resuspended in RNase-free water.

Protocol Steps:
  • Buffering The cells are collected and the pellets are resuspended in 3.6 ml AE Buffer (AE=50mM NaAcetate pH 5.2, 10mM EDTA) and then transferred in 15 ml tubes wit 200 µl 10% SDS (w/v). 2 ml of preheated acid phenol (pH 4.3) are added and the falcon is mixed by vortex.
  • Incubation After 10’ of incubation in 65 °C water bath, the samples are incubated on ice.
  • Centrifugation The samples are centrifugated at 5000 rpm in an tabletop centrifuge.
  • Transferral of supernatants to new tube The supernatants are transferred to a new tube and 2 ml of acid phenol is added to repeat the extraction.
  • Phenol extraction After the centrifugation (10’ at 5000 rpm) the supernatant is transferred into a pre-spun (5’ at 1500 rpm) 50 ml Phase Lock Gel tube (Eppendorf 0032 005.330) and 4 ml of chloroform (Fischer BP1145-1) are added into the falcon.
  • Centrifugation After the spin (10’ at 3,000 rpm), the supernatants are precipitated in RNase-free centrifuge tube by adding 1/10 volume 3 M NaAcetate (pH 5.3) and 2.5 volumes of cold ETOH 100% overnight at -20°C. Centrifugation (30’ at 12000 rpm).
  • Washing The pellets are washed with 70% Ethanol two times.
  • Resuspension of pellets When the pellets are dry, they are resuspended in RNase-free water.
Total RNA Isolation from S. cerevisiae Graph


created over 10 years ago (2 March 2009)    last modified over 7 years ago (28 September 2011)   [ RDF Rdf ]   [ RelFinder Relfinder ]