What's going on with phenol?

Dr. Hiranya S. Roychowdhury hroychow at nmsu.edu
Thu Sep 7 23:04:17 EST 2000


First of all, why are you using "water-extracted" phenol for DNA preps? If
that is what you are doing, the phenol is acidic. It will pull down most of
the DNA with itself, leaving you RNA in the aq. phase (quite elementary,
actually).

Secondly, Why are you bothering with phenol extractions in plasmid preps
anyway? I almost never bother with it and have not had trouble with most
manipulations. If you are using it for sequencing, stick w/ Qia columns if
you can afford them. You write that the yield from Qia columns is not good
enough. I wonder why! How much plasmid do you need and for what purpose?
Usually one gets enough plasmid (of the high copy type) from just a 10-15mL
O/N culture to last a lifetime.


At 11:16 AM 9/7/00 +0100, Chadwick, Brad wrote:
>Recently the mini-preps I've done using alkaline lysis and phenol extraction
>have been giving extremely high OD's, making them look brilliant, until I
>check by restriction digest and agarose gel, to find there's very little
>plasmid DNA actually in there.  MPs prepared by Qiagen columns worked fine,
>but the yield is not good enough for what I want. When I looked into the
>problem with the other ones, it turned out that its the phenol. Even pure
>water extracted with phenol gives and OD thats off the scale, and it seems
>that this contaminant which affects the absorbance at 260nm, is potent
>enough to still make a big difference even after the DNA precipitated and
>the supernatant poured off.  It happens with my brand new bottle of MB grade
>phenol/chloroform/isoamyl alcohol from Sigma and it happens with another
>bottle of old oxidized phenol only. I'm certain it never used to happen when
>I was doing MPs years ago, before Qiagen columns existed, but I always did
>them with 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. 
>
>Brad
>
>
>---
>
>
>
Dr. Hiranya Sankar Roychowdhury
Dept. of Molecular Biology	
PO Box 30001 - 3MLS
New Mexico State University
Las Cruces, NM 88003

Lab: (505) 646 4722
Office: (505) 646 8256
hroychow at nmsu.edu


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