Why I found colony for negative control (contain only double digested vector : one blunt and one sticky end)after transformation in DH5-alfa cells ?

07 July 2018 1 340 Report

Why I found colony for negative control on selection plate (contain only double digested vector : one blunt (by EcoRV) and one sticky (by SalI) end with ligation mixture, no insert, after transformation in DH5-alfa cells ? Is it possible that this vector was ligated?

Prasanth Manohar

It may be due to self ligated ends of the vector. You are using two different restriction digestion enzymes so self-ligation is highly possible but not in the case of single restriction digestion enzyme. You can avoid it by using Gateway cloning strategy.

-Cheers

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