RNA Isolation from Synechocystis

Materials and Reagents

1. Cyanobacterial culture (50 ml) (OD₇₅₀ ~ 2) grown in BG11 medium (Rippka, 1988)
   
2. 95% ethanol for molecular biology (keep at -20 °C)
   
3. 70% ethanol for molecular biology (30 ml of RNAse free water and 70 ml 95% ethanol, keep at -20 °C)
   
4. 70% ethanol for sterilization
5. Phenol BioUltra (for molecular biology, TE-saturated, ~73%) (Sigma-Aldrich, catalog number: 77607 ) (keep at 4 °C)
   
6. 1 M Tris-HCl (Life Technologies, catalog number: 15568-025 )
   
7. 0.5 M EDTA-Na₂ (pH 8.0) (Life Technologies, catalog number: AM9260G )
   
8. RNase-free autoclaved water
   
9. Chloroform (Sigma-Aldrich, catalog number: C2432 ) (keep at 4 °C)
   
10. 10 M lithium chloride solution (Fluka, catalog number: 83268 ) (chill on ice before use)
    
11. RNAse-free DNAse
    
12. Agarose for molecular biology (Sigma-Aldrich, catalog number: A9539 )
    
13. TAE electrophoresis buffer (Sigma-Aldrich, catalog number: T9650 )
    
14. RNA ladder (Life Technologies, Invitrogen^TM, catalog number 15620-016 )
    
15. TE buffer (see Recipes)
    
16. 50/100 TE buffer (see Recipes)
    
17. 10 M LiCl solution in water (see Recipes)
    
18. Cell fix solution (see Recipes)
    

Equipment

1. Saran wrap (any type)
   
2. Autoclave bag (Sigma-Aldrich, catalog number: Z692212 )
   
3. Safe-Lock tubes (2.0 ml) (Eppendorf, catalog number: 0030 120.094 )
   
4. 50 ml plastic conical polypropylene tubes (sterile, nuclease-free, nonpyrogenic, autoclavable) (Life Technologies, catalog number: AM12501 or Corning, Falcon )
   
5. Autoclave
   
6. Refrigerated centrifuge with bucket rotor that fits 50 ml conical tubes (Eppendorf, model: 5804 R and Swing-bucket rotor A-4-44 with 50 ml Falcon adapters)
   
7. Refrigerated microcentrifuge preset to 4 °C (Eppendorf, model: 5415 R )
   
8. Water bath preset to 65 °C
   
9. Subzero refrigerators: -20 °C and -70 °C
   
10. Fume hood
    
11. Ice bath
    
12. Multi tube automated vortex (for example, Micro tube mixer MT-400, Tomy Digital Biology)
    
13. UV-spectrophotometer (preferably, Nanodrop)
    
14. Agarose gel electrophoresis equipment (VWR Mini Gel II Complete Horizontal Electrophoresis System, catalog number: 95043-650)
    Note: Workplace, pipettes, gloves etc. should be cleaned with 70% ethanol before RNA isolation procedures. Conical tubes, Eppendorf tubes, pipette tips should be sterilized by autoclaving in autoclave bags before use.
    

Procedure