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Total RNA extraction from yeast cells LETS Buffer : 500 ml Solution stock
 

Summary: Total RNA extraction from yeast cells
Solution :
LETS Buffer : 500 ml Solution stock
0.01 M Tris-HCl Ó pH 7.5 5 ml 1 M
0.1 M LiCl 10 ml 5 M
0.01 M Na2EDTA Ó 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 Ál of LETS buffer, add 1 eppendorf of clean,
sterile glass beads and add 300 Ál of phenol.
4. Vortex 10 times 30 sec. each, alternate 30 sec on ice.
5. Transfer the liquid in an eppendorf ( 400 Ál)
6. Wash the glass beads as follow and transfer in the eppendorf
- 1 time with 200 Ál LETS
- 1 time with 300 Ál chloroform 4 :1 alcohol-isoamyl mix
7. Spin at 13 000 rpm 3 min. at room t ░C.

  

Source: Abou Elela, Sherif - DÚpartement de Microbiologie et d'Infectiologie, UniversitÚ de Sherbrooke

 

Collections: Biology and Medicine