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cDNA-adapter ligation

Bruno Becker bbecker at iastate.edu
Mon Jul 18 18:22:20 EST 1994


Hi cloning experts,

I am in the process of constructing a cDNA library in lgt11 using kits
from Promega (Riboclone). My question is about the excess of adaptors
needed in the ligation to cDNA to sucessfully prevent the ligation of
cDNAs to each other. The Promega kit uses only a 20-fold excess of
ligatable adapter termini (=40-fold molar excess). I am
concerned that this amount of excess may not be enough to avoid the
generation of hybrid-cDNAs, since several cloning manuals ask for at
least 100-fold molar excess of adaptors in the ligation mixture. 
Does anyone have recommendations on this, or does anyone have practical 
experience with those kits? 
Also, I would appreciate any advice on how to examine isolated cDNA
clones for the presence of such cloning artifacts. I realize that this
can be attempted by probing Northern blots or analyzing overlapping cDNA
clones, but: Are there any new methods out there to do this better (RT-PCR?) 

						Thanks!
-- 
Bruno Becker
Email: bbecker at iastate.edu
Phone (USA): (515)294-1985
Fax (USA): (515)294-4141



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