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Summary: Total RNA extraction from yeast cells
Solution :
LETS Buffer : 500 ml Solution stock
0.01 M Tris-HCl à pH 7.5 5 ml 1 M
0.1 M LiCl 10 ml 5 M
0.01 M Na2EDTA à pH 8.0 10 ml 0.5 M
0.2% SDS 5 ml 20%
DEPC H2O 470 ml
Method :
1. Grow a 50 ml culture until the wanted OD, never freeze the cells.
2. Spin cells at 4000 rpm 5 min., wash the pellet 2 times with 20 ml of LETS
buffer.
3. Resuspend the cells in 300 µl of LETS buffer, add 1 eppendorf of clean,
sterile glass beads and add 300 µl of phenol.
4. Vortex 10 times 30 sec. each, alternate 30 sec on ice.
5. Transfer the liquid in an eppendorf ( 400 µl)
6. Wash the glass beads as follow and transfer in the eppendorf
- 1 time with 200 µl LETS
- 1 time with 300 µl chloroform 4 :1 alcohol-isoamyl mix
7. Spin at 13 000 rpm 3 min. at room t °C.
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