What's going on with phenol?
Bernard Murray, PhD
spam at 127.0.0.1
Thu Sep 7 21:29:21 EST 2000
In article <mantei-F00B4D.15535407092000 at news.ethz.ch>, Ned Mantei
<mantei at cell.biol.ethz.ch> wrote:
> In article
> <C5F1CF1F822FD411922000508B636D0810AB36 at agspsrv24.agric.wa.gov.au>,
> bchadwick at agric.wa.gov.au ("Chadwick, Brad") wrote:
> >Recently the mini-preps I've done using alkaline lysis and phenol
> >have been giving extremely high OD's,...
> >When I looked into the
> >problem with the other ones, it turned out that its the phenol....
> >If somebody has experienced this before with phenol I'd like to find out
> >what causes it and whether its avoidable, and particularly whether any
> >problems were experienced with the DNA in downstream processing, like
> >ligations etc.
> Phenol absorbs in the UV because of the aromatic ring....
> If you want to cleanly remove every last bit of phenol, you should 1)
> aspirate, not just pour off, the ethanol supernatant, 2) spin 5 seconds
> to bring down any liquid that may have been hanging on the walls of the
> tube, then aspirate that, 3) add 1 ml of cold 70% ethanol and repeat
> steps 1 and 2, then 4) repeat step 3.
I use the protocol in which you perform an extraction with plain
chloroform after the phenol/chloroform stage. This greatly helps
to pull phenol from the aqueous layer. If you do this then only
one 70% ethanol wash is needed after precipitation.
Yes, phenol will happily mess up any downstream processing requiring
enzymes. Not only will it denature proteins it will also drop the
pH unless you have good buffering.
> On the other hand, >95% of the A260 of a miniprep is from the RNA. I
> don't think measuring absorbance brings anything with minipreps.
Agreed. Most commercial preps include RNase at the alkaline lysis
stage. For home-made midipreps I perform the crude lysis and
2-propanol cleanup then do an RNase digestion in the reduced
volume (cheaper!) followed by phenol, extraction and optional PEG
Bernard P. Murray, PhD
bpmurray at cgl . ucsf . edu
Department of Cellular & Molecular Pharmacology, UCSF
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