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.
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).
After usuing RNAzol, I found that RNA is not soluable in even 21% Ethanol.
in RNAzol you should add 0.4 ml 75% ethanol to 1ml of supernatant, which the final concentation of ethanol is 21%.
(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.
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
You should have a final salt concentration around 300 mM.