Yeast RNA Miniprep
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Yeast RNA Miniprep
- grow cells to mid-log phase (10-20 ml) (or take samples from time courses, 5-25 ml)
- spin cells, discard supernatant
- wash cells once with 1 ml cold RNA buffer
- freeze pellet in dry ice (and than @ -80 °C)
- thaw cells on ice
- add 100-120 µl ice cold RNA-buffer, resuspend by vortexing
- add 1/2 vol. acid washed glass beads
- vortex at highest level 3 min in cold room or use FastPrep machine (20 sec., level 4.5)
- add 450 µl RNA-buffer-SDS (RT), vortex briefly
- add 450 µl equilibrated Phenol
- vortex at highest level 3 min in cold room
- spin full speed 10 min in cold room
- transfer upper phase to fresh tube (do not take any of the interface !)
- add 300 µl equilibrated Phenol , vortex
- add 300 µl Chloroform , vortex
- spin 2 min , extract upper phase once again with Chloroform
- add 20 µl 4 M NaCl , 1 ml Ethanol
- precipitate 30 min at -20 to -80 °C
- spin full speed 10 min
- wash pellet with 150 µl 70 % Ethanol
- air dry pellet
- resuspend in 30-50 µl H2 O , store at -20 to -80 °C
Buffers:
(All buffer components and stock-solutions, except the Tris-stock-solution, are DEPC treated and autoclaved)
RNA-buffer:
50 mM Tris (HCl) pH 7.4; 100 mM NaCl; 10 mM EDTA
[for 100 ml : 5 ml 1M Tris pH 7.4; 2.5 ml 4 M NaCl; 2 ml 0.5 M EDTA]
RNA-buffer-SDS:
RNA-buffer + 1.3 % SDS
