RNA Isolation (Witman Lab)
1. Grow 500ml (250ml) of cells to mid to late log phase.
2. Harvest cells in IEC (2000 RPM for 5'). Decant supernatant. (resuspend for deflagellation)
3. Vortex the cell pellet to resuspend. (a few mls)
4. Add the cells to 100ml (50ml) of 50 lysis buffer (20mM Tris pH 8.0, 20mM EDTA, 5% SDS, and 1mg/ml (100m g/ml) proteinase K). They lysis buffer should be stirred with a stir bar as the cells are added. We add the cells in a thin stream using a 10ml pipet. As the cells are added to the lysis buffer, the solution will become viscous as the cell material dissolves. You may have to increase the speed of the stir plate as the viscosity increases. Try not to foam the solution as this will inactivate the proteinase K.
5. Incubate the cell mixture without stirring for 4 hours.
6. Add 10ml of 3M NaOAC pH 5.2 and mix.
7. Add 25ml of the above solution to 4 (2) 50ml sterile plastic disposable tubes.
8. Add 25ml of phenol/chloroform (50/50) to each tube and vortex. Separate the phases by centrifugation (2500 RPM 30').
9. Remove the aqueous phase with sterile disposable pipet.
10. Add equal volume of isopropanol and incubate for 15' at room temperature. Collect the RNA by centrifugation (2500 RPM for 10'). Wash the pellet with 80% ETOH.
11. Dissolve the pellet in 5ml of sterile DEPC treated water.
12. Add 5ml of 4M LiCL (DEPC treated)
13. Incubate on ice 16 hours.
14. Collect the RNA by centrifugation (2500 RPM for 30' at 4)
15. The pellet is very soft at this stage so be careful when you decant the supernatant. Try to remove as much of the supernatant as you can as this fraction contains the DNA and carbohydrate.
16. Dissolve the pellet in 3.8ml of sterile DEPC treated water and 0.4ml of 3M NaOAC and 10ml of ETOH. Mix and store at -70. (At this point, combined the 2 pellets and then resuspended in 3.6ml H2O)
17. The RNA in this form is very stable. When you wish to use the RNA vortex the tube and take a measured aliquot. Centrifuge to collect the RNA, wash the pellet with 80% ETOH and dry. The RNA can then be dissolved in water and used for poly A isolation and other purposes.
Lysis Buffer
|
Stock |
Dil. |
150ml |
300ml |
100ml |
|
|
20mM Tris |
1M |
1/50 |
3ml |
6ml |
2ml |
|
20mM Edta |
.5M |
1/25 |
6ml |
12ml |
4ml |
|
5% SDS |
20% |
1/4 |
37.5ml |
75ml |
25ml |
3M NaOAC 40.81g / 100ml
4M LiCl 16.96g / 100ml
Alternate:
1. Grow 250ml cells late log phase / sample i.e 250 control, 250 deflagellation.
2. Pellet cells and resuspend in 100ml fresh Tap for deflagellation. Deflagellate using pH stock - then stir in bright light for 1 hour.
3. Harvest cells IGC 2000 rpm 5'. Decant supernatant and vortex cell pellet to resuspend. Add cells to 50ml lysis buffer while stirring buffer (remember 1mg/ml proteinase K). Buffer will be viscous so stir hard without foaming.
4. Incubate without stirring for 4 hours. Add 10ml 3M NaOAC pH 5.2 and mix.
5. Add 25ml of above to 2 50ml sterile pasteur disposable tubes. Add 25ml pH/chl to each and vortex. Centrifuge at 2500 rpm for 30'.
6. Remove aqueous phase with sterile disposable pipet. Add equal volume isoproponal and incubate 15' at room temperature. Collect RNA by spinning at 2500 rpm for 10'. Wash pellet with 80% ETOH.
7. Dissolve in 5ml DEPC H2O. Add 5ml 4M LiCl DEPC treated. Incubate on ice for 16 hours. Collect by centrifugation at 2500 for 30' at 4P. Pellet will be soft - remove as much supernatant as possible.
8. Dissolve 3.6ml water and add 0.4ml 3M NaOAC, 10ml ETOH. Mix and store at -70. When you want to use, vortex and take a measured aliquot. Centrifuge and wash pellet with 80% ETOH and dry. Dissolve in water.