22 replies to this topic

[mario2004]

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

[methylnick]

mario2004, on Mar 13 2005, 10:35 AM, said:

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?

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

[mario2004]

Thanks Nick. Very helpful.

I still want to know how much ethanol should be used for washing.

Mario

[fred_33]

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

[badcell]

According to Ambion (Tips for handling RNA)

Quote

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.

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!

[methylnick]

Thanks for the great info Badcell,

FYI, I use 70% ethanol for my DNA washes.

Nick

[lula]

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

[bioforum]

Full version of this thread can be found here http://www.protocol-...posts/5564.html

[hobglobin]

methylnick, on Mar 14 2005, 05:30 AM, said:

mario2004, on Mar 13 2005, 10:35 AM, said:

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?

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

Topic 2: I'd avoid to vortex the tube, mixing it gently or flip it with the fingers . There was a poll for it.

[methylnick]

hobglobin, on Feb 10 2009, 10:43 AM, said:

Topic 2: I'd avoid to vortex the tube, mixing it gently or flip it with the fingers . There was a poll for it.

I would be interested to know why this is so?

[swanny]

methylnick, on Feb 11 2009, 09:36 AM, said:

hobglobin, on Feb 10 2009, 10:43 AM, said:

Topic 2: I'd avoid to vortex the tube, mixing it gently or flip it with the fingers . There was a poll for it.

I would be interested to know why this is so?

I have heard two reasons.
1. At this stage, you're just trying to wash out salts, so there's no need to be too vigorous.
2. The pellet is actually not as stable as you might think. It could be that the salts are actually helping it hold together (weird, but that's DNA for ya!).
Also, you want to be very careful when you decant the EtOH after the second spin, because the pellet might not stick to the tube as well as it did after the first spin.

[methylnick]

interesting! wonder if it shears it or not?

nick

[hobglobin]

methylnick, on Feb 11 2009, 03:59 AM, said:

interesting! wonder if it shears it or not?

nick

That would have been my reason to do it, but perhaps overcautious. During work with genomic DNA I try to avoid vortexing as far as possible or do it as short as possible (and with reduced speed).

If the pellet sticks to the tube depends also on the tube type (some seem to have a smoother surface and the pellet is quite loosely attached you notice it at the latest in the speedvac, when it's gone   )

[Ecoli0157]

I am surprised that most people seem to voltex DNA during EtOH wash step.

As for the g.DNA prep, I wash with ~500ul of 70% EtOH and then decant ethanol without resuspending DNA.
DNA pellet is very tight at this step and vigorous voltexing might shear g.DNA which may be undesirable.

Just my thought....

[swanny]

I have never been overly concerned about a bit of gDNA shearing in a gDNA prep. You'd be pretty damned unlucky to have the same stretch of DNA sheared in every single strand, assuming you're looking for a PCR template. And if you were looking to make a library, what's one more random cut-site between friends?

On the other hand, I am cautious about shearing gDNA when I do plasmid preps, because I really don't want the hassle of gDNA contamination of my plasmid.