Dear Yogendra

I am old enough to give you this recepy, as I did this solution by myself - along time ago.

First of all, you need the "Chloroform (24+1)" solution which is prepared as follows:

mix 240ml Chloroform with 10ml Isoamylalkohol.

Second, use the following ingredients to make yourself the "Phenol" solution:

Use 40g Phenol (solid), 10g LowTE buffer (10mM Tris HCL pH 8.0, 1mM EDTA pH 8.0) and 50 g of your existing "Chloroform" solution.

Stir it (even by slightly heating) and then filter it into your glas bottle. We always used some staniol paper (wrapped around the bottle to protect against light) and kept in the fridge at 4˚C.

Any further question?

my best

Rolf

Hi Yogendra!

I really thought you are joking! I strongly recommend that you buy a commercial solution of buffered phenol. Preparing a phenol buffered solution is a nasty experience and I thought nobody is doing it. You can find the protocol in Maniatis. I done it once longtime ago and I can hardly recall the protocol but in summary, you will have to mix an equal volume of liquid phenol

(if you have solid phenol you can heat it at 50 oC) with an equal volume of 0.5M or 1M Tris pH 7.5 or 8, stir at RT in a fumehood for 30 min or more depending on the volume, let settle and decant to separate the organic phase, and repeat until the pH of the phenol reaches the pH of the buffer. Check the pH using paper pH strips. Don’t use electrodes. Do a couple of final mixes with 10 mM Tris pH 7.5 or 8 decant and store it in a dark bottle at 4 oC. Then you can prepare your 25:24:1 solution from this stock. As you can see, it may be more expensive if you prepare it yourself than if you buy it. Good luck!

Thanks for your kind reply..... I think i should buy it

you will get the Phenol/CHCl3/Isoamyl Alcohol (25:24:1, v/v) solution commercially.

We procure it from sigma for DNA extraction.

i have included the wbsite link below.

http://www.sigmaaldrich.com/catalog/search?interface=All&term=Phenol/chloroform/Isoamyl%20Alcohol%20&lang=en®ion=IN&focus=product&N=0+220003048+219853147+219853286

I agree with Abdelhalim. Definitely buy the pre-equilibrated phenol. Don't forget to get your hazardous waste station set up in the fume hood for dry and liquid waste. Wear latex or equivalent gloves when handling the stuff. Don't forget to make just the chlorophorm:isoamyl 24:1 solution by its self. The purpose of the chlorophorm is to remove the phenol. Several extractions with chlorophorm are needed. The isoamyl alchohol is there to prevent foaming and thus make it easier to separate layers after centrifugation. At the end of my phenol chlorophorm extractions I dialyze away any residual solvents. Good luck

.

I generally agree with all of you (except for the fact that I´d prefer to leave out assumptions on who is bying or using self-made solutions which I would regard as inapropriate comments on a platform like this). Maniatis is definitely the resourece if you want to do common solutions yourself. But I would like to add, that placing this question into commonly used search-engines, the first hit already is:

http://www.mrc-lmb.cam.ac.uk/ms/methods/phenol.html

I wonder if posting such questions on a forum is quicker than asking your supervisor or typing it into a search engine......

This comment is to be regarded as my personal opinion and is surely not only related to the person having asked this question.

Good luck with your extractions anyway.

How much total volume you want..

Dear Yogendra

I am old enough to give you this recepy, as I did this solution by myself - along time ago.

First of all, you need the "Chloroform (24+1)" solution which is prepared as follows:

mix 240ml Chloroform with 10ml Isoamylalkohol.

Second, use the following ingredients to make yourself the "Phenol" solution:

Use 40g Phenol (solid), 10g LowTE buffer (10mM Tris HCL pH 8.0, 1mM EDTA pH 8.0) and 50 g of your existing "Chloroform" solution.

Stir it (even by slightly heating) and then filter it into your glas bottle. We always used some staniol paper (wrapped around the bottle to protect against light) and kept in the fridge at 4˚C.

Any further question?

my best

Rolf

Below you can find description how prepare phenol saturated with TRIS.

To 400 ml of liquid phenol (heat it at 65 oC) you should add 0.5 g 8-hydroxyquinoline and incubate in water bath (65 oC) for resolving. Next, you have to add half volume of 0.5 M TRIS (base) and mix 2 hours. After stratification you have to discard upper water layer and check pH (should be 7.7-8.0). After this you have to add equal volume 0.4 M Tris-HCl and mix 30 minutes. Next, you have to discard upper layer leaving their 1 cm thick.

Good luck!

Hi Johachim!

I think there is nothing wrong in suggesting to a peer a personal opinion. RG is specially meant for that, especially when considering the hazard associated with phenol. I think this is what Yogendra really wanted to hear because as you said it may be quicker to find a protocol online than posting the question on RG. People should have in mind the Murphy law and how to deal with its consequences.

It also depends on standards implemented on place. It is more appropriate, safe and cheap in this case to buy a ready solution than applying for permission to make it. For example on some research centers in which I have been (Wellcome Trust center for cell biology, University of Edinburgh) you will not be allowed, IN PRINCIPLE, to perform any new potentially risky protocol unless it is approved by the Safety officer. You will have to fill a form for the Safety office where you will have to explain the safety procedures and all the risks associated with handling of such toxic compounds, etc… apart from whether or not the center has the appropriates facilities for the disposal of such toxics wastes. A person has died because he didn’t observe the safety procedure put in place when pouring liquid nitrogen from the container placed in a closed room, a chemist just blown the lab because he didn’t know about the compounds he was handling , etc,…all these experiences are documented. So, I am a bit surprised to hear you say that advising is not appropriate on RG.

I have been from ULB Brussels to Radboud University Nijmegen at the lab of Henk Stunnenberg more than a couple of times for an all-day consortium group meeting and I thought you have the same high standards when it comes to procedures.

Dear Abdelhalim,

ad1) Please read my post again. I am NOT critizising advice (which for RG is undoubtetly designed) but the tone in which advice is posted. No need to discredit people who do not buy solutions - as long as they can prove a proper standard. Opening a post with "I thought you are joking" can be misleading here.

ad2) A reliably self-made solution does not automatically mean to violate local rules and regulation nor does it mean to lower a high standard (which I hope to meet in any facility). How high our standard is, is not on me to comment but on some unprejudiced authority form outside. As long as we get our accreditations, I assume we met the standards you are refering to.

Please feel free to e-mail me directly if you see the need for further discussions, becasue I feel that we are leaving the sunject this topic was initiated for. If you would like to lead this discussion in public, let´s open a new topic.

Dear Joachim!

I sent you a message on RG! I publically apologize to Yogendra if the tone of my response was somewhat inappropriate but preparing phenol makes people feel sick even if prepared in a fumehood.

I don't see the need for a further topic on whether or not to buy or prepare a phenol solution. I personally prefer to prepar my own solutions but not when there is a hazard associated with them in the process.

You want to use phenol for RNA extraction or DNA?? pH will be different for these when you buffer phenol.

First of all, you need the "Chloroform (24+1)" solution which is prepared as follows:

mix 240ml Chloroform with 10ml Isoamylalkohol.

Second, use the following ingredients to make yourself the "Phenol" solution:

Use 40g Phenol (solid), 10g LowTE buffer (10mM Tris HCL pH 8.0, 1mM EDTA pH 8.0) and 50 g of your existing "Chloroform" solution.