Table 3
From: Optimized RNA Extraction and Northern Hybridization in Streptomycetes
Northern RNA protocol |
|---|
1. Grow bacteria on cellophane covered plates. |
2. Harvest mycelium by scraping and promptly freeze it by immersion in liquid N2. |
3. Grind the frozen sample in Mikrodismembrator for 45 s at 2,000 rpm (alternatively, it can be stored at -80°C). |
4. Extract RNA according to [3]. Prewarm SolD and use larger volumes (1.5 ml/100 mg mycelium). |
5. Further purify RNA by ethanol precipitation in the presence of 2.5 M ammonium acetate. |
6. Glyoxylate and fractionate the RNA according to [5]. |
7. Soak the gel in 20 mM NaOH for 5 min in agitation. |
8. Transfer the RNA to a nylon membrane by alkaline downward blotting according to [6]. |
9. Hybridize the filter to an antisense riboprobe. |