Size fractionating cDNA

Dr. S. Bhattacharya sbhattac at crc.ac.uk
Fri Jan 28 04:43:45 EST 1994


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

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Shoumo Bhattacharya		
MRC Molecular Medicine Group
Royal Postgraduate Medical School
Hammersmith Hospital, LondonW12 0NN UK
Tel:081-7435566 ext.2343
Fax:081-7498341
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