Tips on ethanol precipitation of nucleic acids and wash - (Mar/13/2005 )
Hi bioforumers,
I have a question which has been haunting my mind for a few years. About ethanol wash, almost all protocols just tell you to do a ethanol wash after precipitation but don't tell how. My questions are:
1. How much ethanol (70% for DNA, 75% for RNA) should be added?
2. Should I mix or vortex the tube after adding the ethanol?
3. Why is a lower centrifuge speed used for washing compared to the precipitation, Is higher speed OK for washing because I am always tempted to spin at high speed to be sure my DNA will adhere tighter to the wall of the tube.
4. Why 70% is used for DNA and 75% for RNA?
Thank you.
Mario
2. Should I mix or vortex the tube after adding the ethanol?
3. Why is a lower centrifuge speed used for washing compared to the precipitation, Is higher speed OK for washing because I am always tempted to spin at high speed to be sure my DNA will adhere tighter to the wall of the tube.
4. Why 70% is used for DNA and 75% for RNA?
Hi Mario,
I think you are right about the amounts of ethanol for washing your DNA and RNA pellets, I have used 70% for both.
I think the lower percentage still retains the nucleic acid as a precipitate while salts that may be contaminating the isolation will readily dissolve, thus you are able to remove the salts.
2. You can vortex the tube, in fact I think this is reccommended.
3. As with the shorter centrifuge speed, I think it has to do with us scientists not wanting to wait too long
. The DNA/RNA is already as a precipitate and will happily stick to the bottom of the tube. I have read protocols that say to perform a shorter spin but not a lower centrifuge force. After precipitation I wash and spin at maximum for 5 minutes and that usually does the trick.hope this helps you on your way!
Nick
Thanks Nick. Very helpful.
I still want to know how much ethanol should be used for washing.
Mario
hi
depends of the volume of your preparation for the dna precipitation. But there is no precise quantity. So i tell you my "recipe" for information.
up to 1.5ml, i put the same volume of ethanol 70 as the volume of precipitation prep. upper than 1.5ml, i put 2ml in all cases. It works.
Fred
According to Ambion (Tips for handling RNA)
Ethanol washes are performed after salt/EtOH precipitations to remove any residual salt from the nucleic acid pellet. The wash employs 70-80% EtOH which will solubilize salts but not nucleic acids.
Do
Add 70 - 80% EtOH to the nucleic acid pellet. The volume should be sufficient to at least cover the pellet and wet the sides of the tube when vortexed (there is no volume too large). Vortex the sample for 1 minute; the pellet should come loose from the tube and be broken up in the EtOH. Centrifuge the sample 10 - 30 minutes, to recollect the pellet. Aspirate off the EtOH.
Don't
Don't just add the EtOH and immediately decant. The pellet should be vortexed so that the EtOH can penetrate the sample and solubilize salt.
Don't forget to respin! The pellet must be firmly reaffixed to the tube so that it is not lost during aspiration.
I use 70% ethanol for both DNA and RNA. I always add 1 ml 70% Etoh, vortex and spin the sample at 7k rpm on a microfuge for 5 min. In my hands, it works.
Cheers!
Thanks for the great info Badcell,
FYI, I use 70% ethanol for my DNA washes.
Nick
hello
when preparing DNA i wash with 75% ethanol
i centrifuge at the maximum speed
and i repeat the wash twice 
and i get nice results ,,,,,u know for the volume used i just excee the originla volume of blood i used in the first place ( if im starting with 300 blood ,,,then i wash with 500 ethanol ) usually it works well for me
Ethanol Wash
Ethanol washes are performed after salt/EtOH precipitations to remove any residual salt from the nucleic acid pellet. The wash employs 70-80% EtOH which will solubilize salts but not nucleic acids.
I use 70% ethanol for both DNA and RNA. I always add 1 ml 70% Etoh, vortex and spin the sample at 7k rpm on a microfuge for 5 min. In my hands, it works.
Cheers!
hi
regarding the 70% ethanol wash.....i came across one ref (Nucleic acid research Vol 19, No 19, page 5444....Debomoy Lahiri and J Numberger Jr) which say use ice-cold 70% ethanol for washing.....is there any specfic reason for that?....does it help in solubilizing the salts?
swarup
hi
cold ethanol helps pellet DNA better than RT ethanol.
I am using ethanol precipitation to concentrate DNA for subsequent cloning, however, I am having trouble recovering my DNA. I start by adding 1/10 volume of sodium acetate and 2.5X volume of 100% EtOH, vortex, and precipitate at -80. How long should I be doing this?? Should I be able to see the DNA following the time in -80??? I then spin the DNA for 10 minutes and remove the supernatant...what is the best way to remove the supernatant without disturbing the DNA? I then rinse the DNA with 70% EtOH and spin again. Any one have any suggestions to help with the recovery of DNA? Should the EtOH be cold?