I'm gonna carry out genomic DNA extraction from E. coli using PCI method. Is it still acceptable to use PCI with its bottom layer that has already turned slightly orange in colour? Will I still able to get a high yield of DNA out of the bacteria?
Hi Henry,
I would personally discard oxidized PCI and process my sample with a new batch of reactive. It will still work, but oxidized PCI can damage your DNA (nicking) or RNA (degrade) , so use it at your own risk!
Nicolas
If its less expensive to grow more bacteria than purchase more phenol mix, I would re-equilibrate the phenol to Tris buffer (something like 100mM) pH 7.5 after removing as much of the aqueous layer as possible. If your DNA yield is very poor, at least it was worth a try!
I stand corrected, although I wasn't trying to remove such products as I don't know what they are, but trying to ensure that the aquesous phase in the phenol was appropriate fo rDNA extraction. This raises an interesting point about precisely what damage oxidised phenol (a chemical structure of which would be helpful!) can generate in DNA? I ask because I am using phenol I distilled about 20 years ago (kept under N2).
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