jow at helix.nih.gov
Fri Jul 15 13:25:05 EST 1994
In article <303gk6$ilq at nic.umass.edu> Bindu Chawla,
bindu at titan.ucs.umass.edu writes:
>Hi, I have been trying to isolate total RNA from styles of flowers. The
>is that one of the most abundant proteins in the style is RNAse and
>have been getting partially degraded RNA, I have been usung a buffer
>high concentration of urea and b-mercaptoethanol. Also I haven't been
>DEPC water or baking my glassware. This seems to have no effect on leaf
>isolation, though. Any suggestions would be greatly appreciated.
There was a message from lab_winoto at maillink.berkeley.edu two-and-a-half
weeks ago that gave a method you might try. I have not used this method
myself, but it is very similar to the one routinely used in my group with
50 g of guanidinium thiocyanate (Fluka grade)
0.5 g sodium N-lauryl sarcosine (NOT SDS!)
0.83 ml of 3M NaOAc pH 5.2
0.33 g of Antifoam (optional)
Add water to near 100 ml, stir on a warm plate
pH to 7.0 with a few drops of 10M NaOH (~160ul). Be careful not to
Add 0.7 ml of beta-mercaptoethanol and bring volume to 100 ml.
Filter through a 0.45 um filter unit.
Solution should be clear and weigh ~1.1 to 1.2 g/ml
(see Maniatis or "Current Protocols")
1 Mix one volume of phenol with one volume of Guanidinium solution. This
solution is RNase-ALL.
2 Add up to 100 mg of tissue to 2.0 ml of RNase-ALL at room temperature
and immediately homogenize with a motor driven homogenizer. The
homogenization is preferably done in a 10 ml round bottom polypropylene
3 Add 1/10 volume of chloroform:isoamyl alcohol (24:1), vortex for 20
seconds and incubate on ice for 20 minutes. This step is crucial; don't
skip it or change it!
4 Centrifuge at 10, 000xg for at least 15 minutes (longer than 20' is not
5 *Transfer the upper phase to a fresh 10 ml tube and add one volume of
isopropanol. Allow the RNA to precipitate for 1-2 hours at -20o C. Do
not exceed this time frame. If you wish to precipitate overnight, do it
at 4o C.
6 Wash with 80% ethanol and resuspend the RNA pellet in 1.0 ml of
DEPC-treated water. Store at -70o C.
* If the upper phase is tinted or cloudy, transfer the upper phase to a
new tube containing 1.0 ml phenol (not RNase-ALL). The phases will
immediately mix. Add chloroform as above in step 3 and complete steps
Note: The RNA is now suitable for RNase protection, northern analysis
and reverse transcriptase reactions. If you wish to isolate polyA RNA
then an ethanol precipitation must follow step six. Use 1/8 volume 3M
NaOAc pH 6.0 as the counter ion.
Yields are very high and almost invariably contain the full spectrum of
RNAs, including 4S and 5S species.
More information about the Methods