Chlamy RNA Prep

Protocol

1. Grow 250 ml of cells until they are mid to late log phase
2. Pellet the cells in a 250 ml round bottomed centrifuge bottle in the IEC (2.2k rpm for 10 minutes).
3. Decant the supernatant.
4. Vortex the pellet to resuspend the cells.
5. Add the cells to 50 ml of Lysis Buffer. The cells should be added in a thin stream to the buffer while the buffer is being stirred with a stir bar. The solution will become viscous as the cells are added. It may be necessary to increase the stirring speed during the course of the cell addition. Avoid foaming during the addition as this will inactivate the Proteinase K.

   Lysis Buffer (500 ml)
   [Stock]	Reagent	Amount	[Final]
   1 M	Tris pH=8.0	10 ml	20 mM
   0.5 M	EDTA	20 ml	20 mM
   20%	SDS	125 ml	5%
   --	Proteinase K	--	100 µg/ml to 1 mg/ml
   --	H2O	345 ml	--

   Note: The Proteinase K concentration called for in the original protocol is 1 mg/ml, but in our hands 100 µg/ml is sufficient.

6. Incubate, without stirring, for 4 hours.
7. Add 5 ml of 3M Sodium Acetate (pH = 5.2, made with DEPC water) and mix.
8. Divide the solution into 2 50 ml conical tubes.
9. Add 25 ml of Phenol/CHCl₃ to each tube and vortex.
10. Separate the aqueous and organic phases by spinning in the IEC for 15 minutes at 2.2k rpm.
11. Remove the aqueous phase from each tube using a sterile disposable pipet and put in a new 50 ml conical tube.
12. Precipitate the RNA by adding an equal volume of isopropyl alcohol and incubating for 15 minutes at room temperature.
13. Pellet the RNA by spinning in the IEC for 10 minutes at 2.5k rpm.
14. Wash the pellet with 80% ethanol.
15. Dissolve each pellet in 5 ml of DEPC treated water.
16. Add 5 ml of 4 M LiCl (DEPC treated).
17. Incubate on ice for 16 hours.
18. Pellet the RNA in the IEC for 30 minutes at 2.5k rpm.
19. Carefully decant the supernatant. Be aware that the pellets are very soft. Try to remove as much of the supernatant as possible because it contains DNA and carbohydrates.
20. At this point combine the pellets by dissolving in a total of 3.6 ml of DEPC treated water. Add 0.4 ml of 3 M Sodium Acetate and 10 ml of ethanol. Mix well and store at -70°.
21. The RNA in this form is quite stable. When you want to use some of the RNA, vortex the tube and take a measured aliquot, centrifuge to pellet the RNA, wash with 80% ethanol and dry. The RNA can then be dissolved in water and used for poly-A isolation or other purposes.