LiCl RNA Preparation (Ambros Lab)

LiCl RNA Preparation (Ambros Lab)1) Freeze worms by dropping into liquid nitrogen

2) Grind to a fine powder in a mortar that was precooled with liquid nitrogen

3) Transfer powder to a Falcon tube

4) Add an equal volume of 2X LETS (i.e., equal to the original vol. of worms)

5) Add an equal volume of phenol:chloroform (i.e, twice the original vol. of worms)

6) Vortex and shake until powder is completely thawed

7) Separate phases in a clinical centrifuge at 3000 rpm, about 5-10 minutes, room temperature

8) Extract with phenol:chloroform 3x or more until interface is almost completely clear

9) Precipitate by adding LiCL to 0.2 M and 3 volumes of 100% ETOH and put at -20oC for 30 minutes or whatever is convenient (I generally observe a precipitate immediately)

10) Collect precipitate by centrifugation in clinical centrifuge, 3000 rpm, 10-15 minutes room temperature

11) Rinse pellet several times with 70% ETOH and resuspend in DEPC treated water or TE

12) Read OD260/280 on an appropriate dilution: a ratio of 2.0 implies a fairly pure preparation of RNA

13) Examine on a denaturing gel to assess the integrity of the RNA

14) Store RNA as an ethanol precipitate or at -70oC

(The trace amount of DNA contamination can be removed with DNAse, if necessary)

2X LETS 200 mM LiCL 20 mM EDTA 20 mM Tris pH 7.8 2% SDS (I add vanadyl ribonucleoside complexes to 2 mM just before use - from Sigma)