13 replies to this topic

[lucilius]

Dear all, I have the following question: when using my DNA extraction protocol (fenol extraction) I have 2 steps of ethanol purification. In the first step I have to use 100% ethanol and then in the second step I have to use 70% ethanol.

Why is this?

I can understand you need to do it twice to have a better purification, but why first with 100% and then with 70%?

Also: after I added 100% ethanol I have to "incubate" the samples in the freezer (-25) for about a half an hour, but then I have to to the 75% step, and here I only have to centrifuge the samples, not "incubate".

Is the 75% step simple an extra purification step  to get rid of some 100% ethanol and other stuff?

[phage434]

In the first step, you add 100% ethanol to your existing sample.  This forms a 70% solution of ethanol, if the amounts are correct.  In the second step, you have eliminated all of the liquid in your existing sampe, and have a pellet.  The sample is then washed with 70% ethanol, the same concentration of ethanol as in the first step.

There is disagreement on the necessity (or even desirability) of freezing the sample after the first step.  I'm in the camp that does it.

[lucilius]

Oh yes, indeed.

Another question: Why 70% ethanol? Where does the 70% come from?

Can I use 100% or even 60%?

I can imagine that they use 70% because thats the lowest % of ethanol you can use and thus the cheapest, but I have no idea if this is true?

[phage434]

DNA is soluble in water, and insoluble in 70% ethanol (if there is sufficient salt).  Adding 100% ethanol to the solution to form  a 70% solution allows the DNA to precipitate.  In the wash step, you could wash with 100% ethanol, but it would not dissolve the salt from the pellet, whereas the salt in the pellet is largely removed with a 70% ethanol wash.

[lucilius]

I see.

thanks a lot.


Another question: when doing a DNA extraction, people often add RNASE, in my protocol its states to use 0.1ng/µl rnase. What if I would use a bigger amount?
Could it have an influence in the DNA or purity?

[mdfenko]

lucilius, on Apr 2 2010, 11:11 AM, said:

I see.

thanks a lot.


Another question: when doing a DNA extraction, people often add RNASE, in my protocol its states to use 0.1ng/µl rnase. What if I would use a bigger amount?
Could it have an influence in the DNA or purity?

using more rnase will have no influence on dna or purity, it will just clear the rna faster.

[perneseblue]

mdfenko, on Apr 2 2010, 07:52 AM, said:

using more rnase will have no influence on dna or purity, it will just clear the rna faster.

But it will cost more. And may upset the boss.

[PandaCreamPuff]

perneseblue, on Apr 3 2010, 02:12 AM, said:

mdfenko, on Apr 2 2010, 07:52 AM, said:

using more rnase will have no influence on dna or purity, it will just clear the rna faster.

But it will cost more. And may upset the boss.

the main reason   Unless you are self-funded then you'll pay for the rnase yourself >_<

[josse]

I have also a question on DNA purification.

Why do people add Sodium acetate when doing an ethanolprecipitation?

[phage434]

Positive sodium ions are necessary to bind to the phosphate backbone of DNA, neutralizing its negative charge.  This allows strands to coalesce and precipitate, since the strands would otherwise repel due to similar negative charges.  Most any salt will do, but sodium acetate is the default choice.

[josse]

Ah ok,

thanks a lot phage434

[swanny]

josse, on Apr 16 2010, 11:10 PM, said:

I have also a question on DNA purification.

Why do people add Sodium acetate when doing an ethanolprecipitation?

If you look at the details of the old Birnboim and Doly method (aka alkaline lysis) the gDNA and proteins are precipitated with potassium Ac, and there is also a good protocol using ammonium Ac for DNA precipitation, which is good for smaller DNA fragments. I think Na is often used because you need less to precipitate the DNA.

[josse]

ah interesting swanny,

I'll check that

[Adrian K]

ok, now I am a bit confused,

lets say we use salt precipitating method, or commercial kits such as promega wizard genomic DNA purification kit, the common step is using a cell lysis solution (contains ~2% SDS, EDTA Tris), Protein precipitation solution (~7.5M ammonium acetate)...

As was discussed, the sodium or ammmonium ions is to coalesce and precipitate DNA, but when ammonium acetate is added to precipitate proteins, will it be precipitate out the DNA as well? This is because after the ammonium acetate being added, we incubate the sample on ice, then centrifuge down the pellet (which I believe is protein pellet), and transfer the supernatant for DNA precipitation using alcohol.  IF the DNA had been precipitated when ammonium acetate being added, the supernatant we transfer shouldn't contain any DNA.... so how is it actually happen?