Hi Marzieh, I guess your problem is related to DNA extraction from a biological sample. Well, using the phenol / chloroform-isoamyl alchool procedure you should obtain two phases: the upper phase is aqueous and contains the nucleic acids you wish to extract; the lower one is organic and contains RNA and lipids; proteins separate into the organic phase or lie at the phase interface. A general procedure could be as follows.

Note- It is typically easiest to carry the extraction out in 1.7–2 mL eppendorf tubes.

1. Dilute your nucleic acid sample to 100–700 µL or divide your samples into tubes such that you have no more than 700 µL per tube. It is difficult to do the extraction with volumes smaller than 100 µL. The sample can be concentrated again after precipitation.

2. Add an equal volume of phenol to the tube, vortex vigorously to mix the phases.

3. Spin in a microfuge at top speed for 1–2 min to separate the phases.

4. Remove the aqueous phase to a new tube, being careful not to transfer any of the protein at the phase interface.

5. Repeat the phenol extraction two more times.

6. Extract the sample two times with an equal volume of chloroform:isoamyl alcohol to remove any trace phenol.

7. Precipitate the nucleic acid.

Reagents: Phenol equilibrated to pH 7.5 (pH is important, see below)

Chloroform:isoamyl alcohol in a 24:1 ratio

NOTES:

* Equilibrated phenol can typically be purchased from commercial sources. Alternatively, you can equilibrate it yourself. Be advised that this is not a fun procedure to carry out. The pH is important since chromosomal DNA will end up in the phenol phase if the pH is acid (arround pH 5). This fact can be used for RNA extraction but it is not a good idea to use acid phenol to clean your chromosomal DNA from proteins.

* Phenol can undergo oxidation. Oxidized phenol will appear yellow or red in color (instead of clear) when saturated with water or buffer.

* Phenol and chloroform should be used in a hood if possible (they are toxic, so pay attention!!!).

* If you're in a hurry, you can shorten the protocol to two phenol extractions and one chloroform extraction.

The number of repetitions also depends on what kind of sample you have and what you want to do with it.

If you have whole cell extracts you want to use more phenol steps as if you have only one restriction enzyme

to get rid of. In the same way you might not care about residual phenol if you just want so run your DNA or RNA

on a gel so one chlorophorm step is sufficient. Some other reactions you can do with nucleic acids are more sensitive

to phenol so you should use chlorophorm two times.

* There are also commercial sources of phenol and chloroform mixed together and equilibrated. These are also sufficient for extraction, and I would recommend doing at least two extractions if you decide to go this route. Phenol:Chloroform:Isoamyl alcohol usually has an upper layer of buffer saturated water in the bottle. Do not use this buffer layer -- you want the phenol/chloroform layer underneath.

* Be careful to determine which layer is the phenol. The density of pure phenol (unlike phenol:chloroform:isoamyl alcohol) is almost 1.0. Small changes in the density of your water layer (excess salt, e.g.) can lead to layer inversion.

* Removing the lower layer first can make it easier to recover the upper layer

* Recovery can be improved by "washing" the upper layer by adding water, vortexing, recentrifuging, and recovering the water layer again.

I wish these advices will help you!! Bye bye

A phenol-chloroform-isoamylalcohol solution should have two distinct phases.

You please redefine what is question clearly ? if you are expecting us to tell the net result of mixing these solvent --it is unqualified as intelligent question .You

SHOULD BETTER TAKE SMALL AMOUNT OF THESE SOLVENTS , WHICH AR. E NOT COSTLY AND SEE FOR URSELF. OTHER WISE IF THEY SEPARATE OUT IN DISTINCT PHASES -THEN U MAY ASK US WHY...

First you should do, try mising them yourself in test tube...then pls .ask question. Please dont mind.

We expect questions to be directed clearly , so that we can provide you correct advise or suggestions, of which you too will be happy with . otherwise the Forum will be filled with weird undirected . answers and will give you more confusion than solution to your specific problem . Hope you understand me and forgive me for being so frank. WIsh you good luck in your career.

Just needed to say that your question is fine Marzieh, and please, please don't ever go around mixing chemicals you don't know about...

Phoebe you r partially right. In present casenone of the chemicals will go wild. I dont understand what is saturated phenol,pls give exact examples of it. Phase separationwil result beyond athreshold cocn ofphenol n iso amyl alcohol.here the immiscibilty is subject to mole fractions/ ratio of the components.

Two phases, phenol is partially soluble in water phase (containing Tris-Hcl, pH=8 in this case), whereas chlorophorm and isoamyl form one phase.

Hi Marzieh, I guess your problem is related to DNA extraction from a biological sample. Well, using the phenol / chloroform-isoamyl alchool procedure you should obtain two phases: the upper phase is aqueous and contains the nucleic acids you wish to extract; the lower one is organic and contains RNA and lipids; proteins separate into the organic phase or lie at the phase interface. A general procedure could be as follows.

Note- It is typically easiest to carry the extraction out in 1.7–2 mL eppendorf tubes.

1. Dilute your nucleic acid sample to 100–700 µL or divide your samples into tubes such that you have no more than 700 µL per tube. It is difficult to do the extraction with volumes smaller than 100 µL. The sample can be concentrated again after precipitation.

2. Add an equal volume of phenol to the tube, vortex vigorously to mix the phases.

3. Spin in a microfuge at top speed for 1–2 min to separate the phases.

4. Remove the aqueous phase to a new tube, being careful not to transfer any of the protein at the phase interface.

5. Repeat the phenol extraction two more times.

6. Extract the sample two times with an equal volume of chloroform:isoamyl alcohol to remove any trace phenol.

7. Precipitate the nucleic acid.

Reagents: Phenol equilibrated to pH 7.5 (pH is important, see below)

Chloroform:isoamyl alcohol in a 24:1 ratio

NOTES:

* Equilibrated phenol can typically be purchased from commercial sources. Alternatively, you can equilibrate it yourself. Be advised that this is not a fun procedure to carry out. The pH is important since chromosomal DNA will end up in the phenol phase if the pH is acid (arround pH 5). This fact can be used for RNA extraction but it is not a good idea to use acid phenol to clean your chromosomal DNA from proteins.

* Phenol can undergo oxidation. Oxidized phenol will appear yellow or red in color (instead of clear) when saturated with water or buffer.

* Phenol and chloroform should be used in a hood if possible (they are toxic, so pay attention!!!).

* If you're in a hurry, you can shorten the protocol to two phenol extractions and one chloroform extraction.

The number of repetitions also depends on what kind of sample you have and what you want to do with it.

If you have whole cell extracts you want to use more phenol steps as if you have only one restriction enzyme

to get rid of. In the same way you might not care about residual phenol if you just want so run your DNA or RNA

on a gel so one chlorophorm step is sufficient. Some other reactions you can do with nucleic acids are more sensitive

to phenol so you should use chlorophorm two times.

* There are also commercial sources of phenol and chloroform mixed together and equilibrated. These are also sufficient for extraction, and I would recommend doing at least two extractions if you decide to go this route. Phenol:Chloroform:Isoamyl alcohol usually has an upper layer of buffer saturated water in the bottle. Do not use this buffer layer -- you want the phenol/chloroform layer underneath.

* Be careful to determine which layer is the phenol. The density of pure phenol (unlike phenol:chloroform:isoamyl alcohol) is almost 1.0. Small changes in the density of your water layer (excess salt, e.g.) can lead to layer inversion.

* Removing the lower layer first can make it easier to recover the upper layer

* Recovery can be improved by "washing" the upper layer by adding water, vortexing, recentrifuging, and recovering the water layer again.

I wish these advices will help you!! Bye bye

Just to say

what a very detailed reply by Graziano Zannini . So, thank you very much Graziano.

Dear, Graziano,

I appreciate your patience, in replying to the Question, with very clear concepts. Thank you

Not at all, ResearchGate also serves this purpose. Doesn't it!? I wish you all nice research!!!

Agree w/ Mohammad and Srinath

That's the beauty of ResearchGate. It helps to build bridges between geographically separated researchers who are willing to collaborate with each others.

Thank you Graziano!

Dear, Graziano,

Thank for your good comments. Indeed i made equilibrated phenol by Tris-HCL 0.5 M, PH:8. Finally, i added chloroform-isoamyl alcohol in a 24:1 ratio to equilibrated phenol for applying in DNA extraction. I prospected that it changes to two phase: upper: chloroform and downer: saturated phenol! but these two aqueous combined. Can i use this combination( phenol-chloroform-isoamyl alcohol) for DNA extraction?

thanks a lot

Dear Marzieh, yes definitely! But I do not agree with your expectation: by adding the phenol:chloroform:isoamyl alcohol (25:24:1) mixture to your sample you will get two phases, of course, but also one interface between them: the aqueous top phase contains the majority of DNA, interface mostly proteins denatured by phenol (appearing as an opaque disc), and lower organic phase most of the lipids (in chloroform; NOTE: chloroform has the highest density, not phenol). Your goal is to collect the upper aqueous phase with the lowest phenol amount as possible (contamination) !!! So: 1. mix equal volume of phenol: chloroform : isoamyl alcohol solution with the sample solution in a eppendorf tube. Close the tube and mix briefly until an emulsion forms. 2. Then centrifuge at 12,000 g for 3-5 minutes. The aqueous phase (upper) and organic phase (lower) should be well separated. 3. Now, transfer the aqueous phase to a new tube and discard the interface and organic phase. Repeat these three steps until no protein is visible at the interface. As optional step to remove any residual phenol, you can mix briefly equal volume of chloroform with the aqueous phase and centrifuge at 12,000 g for 3-5 minutes. I hope these explanations will be of help and good luck with your DNA extraction!

P.S.: You can also take a look in this book: Sambrook, Fritsch, Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Edition, Vol. 3, pages E3 -E4; Cold Spring Harbor Laboratory Press 1989. It is quite old but still fundamental. Believe me!

So thanks Graziano. I will apply your comments and explanations in DNA extraction.

good luck

best

We know that at lower pH after Phenol chloroform extraction RNA remain in aqueous phase and DNA at organic phase. But when we perform the same with Tris saturated phenol (Higher pH) DNA comes to aqueous phase. Can anyone tell me the reason behind it........

Dear Rakesh, as you can guess, the reason behind the phenomenon that you described resides in the different solubility of the two types of nucleic acid in different pH conditions due to their different molecular structure: single-stranded (RNA) vs. double-strand (DNA). Let me explain better. In phenol chloroform extraction, the pH of phenol determines the partitioning of DNA and RNA between the organic and the aqueous phases. Acidic phenol (pH 4-5) retains RNA in the aqueous, but pushes DNA into the organic (phenol) phase because the negatively charged DNA phosphate groups are more easily neutralized (by protonation) than those in RNA: as a matter of fact DNA is less acidic / has a greater pKa than RNA. In this case, DNA dissolves in the organic phase following the rule “like dissolves like”. RNA, on the other hand, is not neutralized in acidic environment because, even though it has negatively charged phosphate groups, it also has its nitrogenous bases exposed (it is single-stranded), which can form hydrogen bonds with water, keeping it in the aqueous phase. At higher (neutral or slightly alkaline) pH (7-8), the phosphate diesters in nucleic acids are, again, negatively charged, and thus DNA and RNA both partition into the aqueous phase. I hope this explanation is sufficiently clear.

Hi everyone,

So I had the same question regarding the resulting upper and lower phases of the phenol-chlorofom-isoamyl solution (prior to mixing with DNA-RNA) and came across two different answers; the one provided here, where Graziano kindly suggests that the upper phase of the solution be discarded prior to use, and an older thread here:

http://boards.straightdope.com/sdmb/showthread.php?t=89419

where it is stated that best is to use the upper phase and the lower phase by vortexing prior to use.

I am curious as to if anyone knows the explicit answer to this... Currently I am discarding the upper phase before using it for the RNA cleaning stage with Phase-Lock gel tubes.

Thanks for your input!

Carmen

thanks Zannini for a clear explanation

so what's the ratio of phenol :chloroform for RNA extraction ,1:1 or 5:1,seems they both work,but which one is better?