RNA isolation protocol for Nostoc punctiforme                                                                        5/2000

1.  Harvest culture and suspend in 0.5 ml of media or water.

2.  Place the 0.5 ml in a MiniBead Beater sterile screw capped tube.  Freeze quickly in liquid nitrogen.  
Cells can be stored at -80oC at this point until all the samples have been collected and are ready to be
processed.

3.  Place tubes on ice and add the following in the order listed:
          0.58 g 0.5 mm glass or zirconium beads (autoclaved)
          33.3 ul 20 % SDS
          167ul 3% Celite (autoclaved)
          583 ul Tris-buffered phenol

Shake in the MiniBead Beater on high setting for 160 sec.  Store on ice until all the samples are ready for 
the next step.

4.  Centrifuge for 15 min at +4C in a microfuge.

5.  Pipette off 600 ul of the top aqueous layer into a new tube and add 36.7 ul 3M sodium acetate and 478 ul 

5% ethanol.  Mix and place at -20C for at least four h or overnight.

6.  Centrifuge samples for 15 min at +4C.

7.  Dissolve the pellet in 250 ul TE + 0.2% SDS.

8.  LiCl precipitation of RNA for removal of DNA:
           a.  Add 62.5 ul of 10M LiCl while vortexing.  Final LiCl concentration is 2M.  Incubate on ice overnight.
  
           b.  Centrifuge samples at +4C for 15 min.

           c.  Rinse RNA pellet with 0.5 ml 2M LiCl; make sure to break up the pellet with the pipette tip.  
           Centrifuge as before.

           d.  Dissolve the pellet in 0.5 ml DEPC treated ddw.  Add 32 ul 3M sodium acetate, 400 ul 
           95% ethanol, and mix.  Place samples at -80C for 20 min.  Centrifuge as before.

           e.  Dissolve the pellet in 50 ul TE+ 0.2% SDS.

9.    Assay by A260 on a spectrophotometer;  1 A260 = 40 ug/ml.

References:  Schmidt-Goff and Federspeil. 1993. J. Bacteriology 175:1806-1813.   Summers et al. 1995. 
          J. Bacteriology 177:6184-6194.