Size fractionation of DNA

John Brennand john.brennand at
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.

Good luck


More information about the Methods mailing list