Hi!
It is very tedious procedure, you have to do very carefully n patiently takes lot of time. I believe your lab has "Molecular Cloning, a laboratory manual" Sambrook et al. edition go through that you get A-Z information otherwise take a look at the following link.
http://ieg.ou.edu/protocol/Protocols/Phenol%20Saturation.pdf
My suggestion if you can afford rather than making waste of your time buy a Sigma Aldrich Tris equilibrated Phenol 500 ml bottle, work very good. Your time would be saved.
Good Luck!!
Which buffer? I would really suggest buying the product ready to use, since working with Phenol is quite hazardous. If you really want to make it yourself, look here: http://www.mrc-lmb.cam.ac.uk/ms/methods/phenol.html
@ Christian -> Basic Phenol for DNA , acidic for RNA -> Basic has to be made with buffer
Dr jetty hydroxyquinoline is not available for me , is it essential?
Hi!
When it is mentioned in protocol I believe you should go according to the procedure. When I was India one of the technician used to do all these buffers very carefully by following Sambrook et al. Manuel, he used to tell you should do very carefully as per the procedure. After coming to Europe the labs where I worked used to order Sigma Tris equilibrated Phenol.
If you want to make the buffer yes you have to use that. Otherwise if you want to take risk try as you like. That was the reason I told why all these risks if you can afford go for Sigma. Choice is yours.
Good Luck!!!
Dr merine I did it today about 6 times to mix with tris buffer the ph of phenol become 8 but the phenol become orange to brown colour ????
How to know if its good or not whats the ideal characters of it???
1st ididnt add 8hydroxy quinoline
2nd i use analytical grade phenol not molecular biology grade
the phenol I just openned the bottle before use (100 gm)
actualy I dunna know if it contain stabilizer or not
@christian is it a must to use molecular grade phenol or analytical grade is ok ?
@Shaimaa: That should be ok. When the bottle is fresh, it can come from the stabilizer (if any is present).
@ all why order it from Sigma for the triple or whatever price... You can get rather cheap equilibrated phenol from every "no name" chemical supply and for the topic of RNASE and DNASE free phenol.. This is a hoax to get more money... What you do when extract with phenol is getting the proteins from your cell in the organic = phenol/chloroform phase and your nucleic acids in the aqueous phase.. So as long as you have a cell lysate it doesn't matter if your chemicals are nuclease free because that is what you do with phenol -> getting the nucleases from your cells out. The cells are full of them!!.. When you precipitate your nucleic acids and then resolve them that is the time for nuclease free reagents and equipment !!!
Hi! In India our Technicians prepare very carefully as per the protocol given in Sambrook et al. I/We never buy. After coming to Europe I saw in the labs of different countries ordering Sigma so advised. Of course cost is high but some chemicals are worth reliable.
@Norbert: Probably because out of ordering habit and because people don't know. The thing with the certified DNAse/RNAse free reagent is something I had once with a representative of a company who praised his Trizol derivative as certified DNAse/RNAse free. He looked a bit puzzled when I told him that I would rather research an enzyme which survives the treatment with phenolic solutions...
First you have to equilibrate phenol with Tris as per Sambrook et a. Then you can make the mix with Chloroform and IAA
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