Phenol/CHCl3 rendering dsDNA single-stranded

R.N. Leach BMBRNL at leeds.ac.uk
Tue Dec 1 13:33:38 EST 1998


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
U.K.



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