RNA from Neurospora

[Nora Plesofsky-Vig]

To Martin Hoyt:

We have developed a procedure for isolating RNA from Neurospora that  
is quick and appears clean, but we have not tested the RNA in  
protection studies:

RNA Extraction Buffer
	8 M Guanidine HCl (practical grade)
	50 mM Tris, pH 7.5
	1 mM EDTA
	8% v/v 2-mercaptoethanol
	0.5% N-lauroylsarcosine
		filter sterilize (0.45 uM filter), store 4oC

1. Homogenize c. 200 mg cells for 60 sec (we use MSK, with cooling  
and 2.7 g glass beads for homogenization) in either one or two ml  
(larger vol throughout for spores) of RNA extraction buffer.

2. Put homogenate in Eppendorf tubes; wash beads with 200 or 400 ul  
of extraction buffer and add to tube(s). Leave tubes at room temp for  
5 min.

3. Spin in microfuge (15,000 x g) for 10 min at 4oC. Collect  
supernatant and spin again.

4. To second supernatant (600-800 ul), add 1/3 vol of ddwater and 1/3  
vol of 10 M LiCl. The final concentration should be 2 M LiCl and 4.8  
M Guanidine HCl.

5. Ppt. RNA overnight at 4oC.

6. To recover ppt RNA, spin in microfuge at 4oC for 10 min, wash  
pellet 3x with 300 ul of 2 M LiCl and 3x with 300 ul of (-20oC) 95%  
ethanol

7. Dry pellet in speed vac and dissolve in DEPC-treated ddwater  
(50-100 ul). RNA pellets dissolve within 15 min at room temp. Clean  
RNA of insoluble material by brief spin, transferring supernatant to  
clean tube. Store RNA at -80oC.

Nora Plesofsky-Vig