phenol turns pink after adding it to the sample - phenol:chloroform DNA extraction (Nov/07/2008 )
Hi there,
I was wondering if you guys have a possible explanation why phenol turns pink after it is added. I am pretty clueless right now.
I am trying to do cell lysis, phenol:chloroform extraction and DNA precipitation.....but when I get to the step were I have to add an equal volume of phenol:chloroform:isoamyl alcohol, my solution turns pick. I checked the Phenol:chloroform:alcohol stock solution and it is colorless. The color didn't change until after it was added to the sample. Do you guy have any ideas what is happening and what I could do?
Cheers!
I was wondering if you guys have a possible explanation why phenol turns pink after it is added. I am pretty clueless right now.
I am trying to do cell lysis, phenol:chloroform extraction and DNA precipitation.....but when I get to the step were I have to add an equal volume of phenol:chloroform:isoamyl alcohol, my solution turns pick. I checked the Phenol:chloroform:alcohol stock solution and it is colorless. The color didn't change until after it was added to the sample. Do you guy have any ideas what is happening and what I could do?
Cheers!
Normally pink or orange is an indication for oxidation of the phenol (and that you shouldn't use it anymore). Also possible: Pigments/colour left over from the tissue and now become soluble?
Thanks for your reply!
I have used a soil sample for extraction. Any idea what kind of pigments could do that?
Could something in the sample oxidize the phenol after it was added? Would role would pH play in the oxidation?
Agree with hobglobin, phenol might be oxidized. Could be component in the soil that is causing the oxidation. As phenol is buffered, don't think pH will change drastically (or you could increase buffering capacity)?
Have you added reducing agent in the DNA isolation step? As the phenol is colorless, I don't think hydroxyquinoline was added. There's a protocol for soil DNA isolation. The author added Dithiothreitol (DTT) as reducing agent prior to phenol-chloroform isolation. That could really helped to minimize oxidants present in the soil... the protocol could be acquired here
http://www.eeescience.utoledo.edu/Faculty/...%20-%20soil.pdf
Bests,
U could opt for using kit to extract DNA from soil i believe there's a lot of kits outside nowadays. Omega biotek got one. Mo Bio i have heard about them too.
if still having problem with extraction
go for --> STOOL KIT.
Have you added reducing agent in the DNA isolation step? As the phenol is colorless, I don't think hydroxyquinoline was added. There's a protocol for soil DNA isolation. The author added Dithiothreitol (DTT) as reducing agent prior to phenol-chloroform isolation. That could really helped to minimize oxidants present in the soil... the protocol could be acquired here
http://www.eeescience.utoledo.edu/Faculty/...%20-%20soil.pdf
Bests,
Thanks guys!
I'll give it a try. Cheers.
if still having problem with extraction
go for --> STOOL KIT.
Hi Hanming86,
was thinking about comparing the DNA extraction with a soil kit, but I wasn't sure how efficient it would be. I guess, I am at this point now where it's definitely worth a try. Cheers!