Yeast RNA Miniprep

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

 

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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