Size fractionating cDNA

[Fred Boyd]

BRL uses a sizing column in their cDNA library kits.  They are pretty well
calibrated and available separately.  We routinely size fractionate out
<500 bp, but it depends on the size of the cDNA you're looking for.  But if
we knew that... ;-)  

If we found what we were looking for the first time it would just be
search, not re-search.


> Hi netters
> 
> What do you think is the best way to size fractionate cDNA
> before cloning?  I am constructing an oligodT primed directional
> expression library, and therefore would like to remove cDNA less
> than 1000 nt.  Is this a reasonable figure to aim for? Or is it
> better to divide it into fractions e.g. >2-3 kb, and from 1-2 kb 
> cloning these separately. 
> 
> To my (novice) mind it seems that running out on agarose
> and electroeluting it seems reasonable and easy, but
> are other methods (sucrose gradient or KOAc gradient) better?
> 
> Also, although it would appear obvious that size fractionation
> would remove unligated linkers is this assumption correct, or is
> it safer to have another linker removing step i.e. sephacryl
> S-400 column.
> 
> Many thanks, and I will post a summary.
> 
> 
> Shoumo
> 
> -------------------------------------------------------
> Shoumo Bhattacharya		
> MRC Molecular Medicine Group
> Royal Postgraduate Medical School
> Hammersmith Hospital, LondonW12 0NN UK
> Tel:081-7435566 ext.2343
> Fax:081-7498341
> -------------------------------------------------------

-- 
Fred Boyd                         Voice- 612-624-8150
Lab. Med. Path.                   Fax- 612-616-2444
Cell Biol. Neuro.                 Lab- 612-624-8154
Box 609, UMHC   
University of Minnesota           E-Mail  fredboyd at bmec.micro.umn.edu
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