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cloning - (Dec/10/2007 )

my concern is..
how to prevent insertion of more than one insert, digested with same RE (e.g EcoRI) during cloning into vector (eg pGEMT easy).
as one insert as well as 2 or 3 inserts also show white colonies in Blue-white Screening method.


This should not be a problem with low concentrations of vector and insert, and equimolar amounts. Try 20 ng of vector and equimolar insert in a ligation. The bimolecular reaction should be strongly favored over the trimolecular reaction.