Phenol/CHCl3 rendering dsDNA single-stranded
s.craig at student.unsw.edu.au
Wed Dec 2 08:29:52 EST 1998
If you want to do away with the phenol/chloroform perhaps you could use
temperature sensitive alkaline phosphatase (Gibco/Life Technologies). I have used
it a few times and it seems to work quite well, the only disadvantage is the
restriction on how much of the dephos reaction can be used in the ligation (due
to EDTA being in the inactivation buffer). It has always done the job in my
Dept. of Biotechnology
University of New South Wales
"R.N. Leach" wrote:
> Recently I've been seeing a strange phenomenon in my plasmid DNA preps.
> I cut my plasmid, gel purify it by cutting the appropriate band out of an
> agarose gel and eluting the DNA by centrifugation. After EtOH/NaoAc
> precipitation and resuspension I dephosphorylate and then phenol/chloroform
> extract the cut, dephosphorylated plasmid DNA ready to ligate in my insert.
> However, when I check a proportion of the phenol/chloroform extracted plasmid
> on a gel I see two bands. It is not uncut plasmid as the same thing happens
> to my PCR products and a vector of a different size, the expected product and
> a product half the size.
> I've narrowed it down to the phenol/chloroform step. I don't heat inactivate
> prior to the extraction (only if I'm feeling really paranoid) but I do
> vigorously vortex my phenol/chloroform+DNA suspensions. I don't believe for a
> moment that mechanical shearage is what I'm seeing so what is happening?
> Logically, I'm going to try a colleague's stock of phenol/chloroform but
> ideally I'd like to do away with the step altogether. Would gel purification
> be sufficient to remove contaminating phosphatases and dephosphatases that
> could creep into ligation reactions?
> Any ideas/information/tips gratefully received.
> Rob Leach, Ph.D.,
> School of Biochemistry & Molecular Biology,
> Uinversity of Leeds,
> LEEDS, LS2 9JT
> W. Yorks., England
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