Chewbby

Total RNA Extraction from Yeast

(discontinued since 2012)


Preparation :


Ensure you have RNAse-free 100% Ethanol at -20°C.
Same for cold RNAse-free 70% Ethanol.
65°C water bath
Bring phenol to room temperature
Fetch some liquid nitrogen (### Wear eye protection and gloves ###)

TES Lysis Buffer:
10 mM Tris-HCl (pH7.4)
10 mM EDTA
0.5 % SDS
IMPORTANT: 
Work under FUME HOOD with eye protection and gloves when handling phenol or chloroform.
Work in RNA dedicated area for the other steps.
Use RNAse-free pipets and tips all the way through.

Extraction:


Harvest 2ml of cell culture at 10e7 cells/ml.
Spin 13,000 rpm for 1min and discard supernatant.
Add 700μl of TES buffer, Vortex and snap-freeze in liquid nitrogen
Add 700μl of room-temperature phenol. Thaw and vortex.
Incubate at 65°C for 20 minutes, vortex every 5-7 minutes during this time.
Snap-freeze in liquid nitrogen for 1 min (or on dry ice for 5min). Thaw and vortex.
Spin at 13,000 rpm for 5min at room temp.
Transfer the upper aqueous phase to a fresh tube.

Add 700μl of room-temperature phenol, vortex well.
Spin at 13,000 rpm for 5min at room temp.
Transfer the upper aqueous phase to a fresh tube.

Add 700μl of chloroform, vortex.
Spin at 13,000 rpm for 5min at room temp.
Transfer the upper aqueous phase to a fresh tube.

Run the sample through an RNeasy mini-column from Qiagen (see Rneasy manual).
Elute in 50 μl  RNAse-free water

Precipitate :
Add 50 μl of 3M NaAcetate and 1.25ml of RNAse-free cold 100% Ethanol, vortex and keep at -20°C for at least 30 minutes.
Spin 13,000 rpm for 5 min at room temp, discard supernatant.
Add 200 μl of RNAse-free cold 70% Ethanol. Dislodge pellet.
Spin 13,000 rpm for 5 min at room temp. Discard supernatant. (be careful not to lose the pellet !)
Let dry for a few minutes (not more) and resuspend in 20-50 μl of Rnase-free water.
Dernière mise à jour : ( 20-01-2009 )