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RNA pellet gone in the wrong concentration of ethanol - (Nov/06/2012 )

When I exacted the RNA from tissue using trizol, I made a mistake after I got the RNA pellet.
I wanted to wash the pellet with 75% ethanol, but unfortunately, the concentration of the ethanol I prepared is 25%.
Then I votex the RNA pellet in 25% ethanol and did the centrifugation.I found that the pellet has gone away. And I add more ethanol high up to the right concentration. After that I did the centrifugation another time.
But still, I can not spin the RNA down to the bottom. Should I add NaCl or other salt to solve this problem? Do I need to incubate it in lower temperture? Is the carrier needed this time?
Really need some help.

-cckishi-

You would need to add salt to get the RNA to re-precipitate. Depending on the concentration of the RNA in the solution you have now you may need the carrier, the lower the concentration, the more lkely you are to need a carrier. Precipitating at -20 won't hurt (make sure it won't freeze/thaw).

-bob1-

After usuing RNAzol, I found that RNA is not soluable in even 21% Ethanol.
http://www.mrcgene.com/rnazol.htm

in RNAzol you should add 0.4 ml 75% ethanol to 1ml of supernatant, which the final concentation of ethanol is 21%.

Calcualation:

(400 ul x 75%) + (1000 ul x 0%) = 1400 ul x X% => X= 21.42 %

So something else is wrong.
One of them:
If the starting cells are less than 1Million, the RNA pellete is very tiny. and specially when RNA is clean, it is very dificult to see it in vial.

I have not used but you can use glycoblue or something else.

-----
Babak Memari

-memari-

Thank you for help.
Do you have the protocol which I should follow? I don't know how much salt should add in to the solution

-cckishi-

You should have a final salt concentration around 300 mM.

-phage434-