Phage DNA isolation

Jacquelyn Clark JacquiBrian at worldnet.att.net
Thu Sep 30 08:12:52 EST 1999


Bob:

There are many ways to skin a cat, but here's how the typical lambda DNA
isolation from lysate works:

First, insoluble cellular debris and gobs of agar get pelleted, then DNase +
RNase treatment removes cellular nucleic acids (Mg++ stabilizes phage, and
is reqd. for DNase activity).  The phage DNA is protected, since it is
enclosed within the phage particle.

Addition of EDTA stops DNase, and simultaneously destabilizes phage
particle, releasing phage DNA into solution

Phenol removes protein to the aqueous/organic interface, and the subsequent
chloroform removes the phenol.  At this point, there is not much left except
phage DNA to be precipitated

EtOH - If you don't do the DNase/RNase treatment before you pop the phage,
EtOH/salt precipitates DNA+RNA almost equally well.  But mainly what you'll
see on a gel is a huge glob of bacterial rRNA.  EtOH alone precipitates
nucleic acid rather poorly by itself, but in the presence of salt efficiency
is improved dramatically (Biophysical explanation someone please!).  But
since everything else in now nucleotides and short oligos, all you get in
the pellet is phage DNA (and a little carbohydrate that shouldn't affect
anything)

Brian





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