Size fractionation of DNA
john.brennand at gbapr.zeneca.com
Thu Aug 15 10:27:03 EST 1996
We have used HPLC (using Waters GenpakFax columns) to do this sort
of separation (& plasmid preps) very efficiently and cleanly.
However it was tiresome to do particularly if you are not a regular
HPLC user - hence everyone in our labs subsequently drifted back to
what they know and love, i.e., using (LMP) agarose gels !
You can also use some of the spin columns that are available from
the usual manafacturers ( check out:- Pharmacia, Clontech, 5',3'
Inc. Amicon, Flowgen) with various size exclusion limits, for this
purpose and designed particularly for cDNA size selection prior to
library construction where the small stuff <500 bp stays in the
column whilst the bigger DNA comes through.
We found that the ones we tried worked ok, but not surprisingly,
weren't 100% efficient - hence we usually put our cDNA through two
runs. If you actually want the 400 bp DNA, I don't know how easy it
is to recover it from these columns.
I'm sure that there are some resins out there that you could use in
conventional column chromatography (or home made spin columns) to
achieve the separation you require (ask the companies) but these
will be an anathema to folks who love their agarose.
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