RNA from Neurospora
npv at MANI.CBS.UMN.EDU
Mon Mar 13 13:14:17 EST 1995
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
RNA Extraction Buffer
8 M Guanidine HCl (practical grade)
50 mM Tris, pH 7.5
1 mM EDTA
8% v/v 2-mercaptoethanol
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
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%
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.
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