Size fractionation of DNA

ProZyme, Inc. info at prozyme.com
Fri Aug 16 20:30:45 EST 1996


Michael Feldman <FeldmanM at mail.med.upenn.edu> wrote:

>I am using a 16 kb immunoglobulin expression vector that contains a 
>400 bp immunoglobulin gene insert. I would like to seprate the 16 kb 
>and 400 bp fragments from each other following restriction digestion. 
>I know this can be done using agarose gel electrophoresis but I am 
>looking for an alternative technique. Any suggestions?

You might try polyethylene glycol precipitation, which will leave
fragments smaller than about 500 bp in the sup while bringing down
larger stuff.  Add 20% PEG 8000/2.5 M NaCl to final concs. of 5% PEG
and 625 mM NaCl.  Incubate on ice for several hours (I'm not sure what
the minimum time is) and microfuge in the cold.  The pellet can be
dissolved in TE.  It would probably be best to phenol extract and EtOH
precipitate before using the DNA.  The same is true for the small
fragment in the sup, though I don't know if you'd have to dialyze away
the PEG first.

This size difference is so great that you might be able to get away
with simple gel filtration if you use a matrix with a really high
exclusion limit.

Good luck!
Bruce Amsden




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