EcoRI cloning giving only inverted inserts!!??
Posted 30 September 2010 - 02:12 PM
To verify the orientation of the insert I´m using an enzyme that cuts one time in the vector and one time in the insert. The difference is very clear (400pb vs 1200pb).
Someone has a clue? Or am I very unlucky?
Posted 30 September 2010 - 02:21 PM
Posted 30 September 2010 - 02:30 PM
Very likely the insert is somewhat or very toxic to the E. coli cell in one of the orientations. Your cloning vector may have a promoter active aimed into the cloning site. You could try a different vector with terminators surrounding the cloning site, or add your own to the sequence. Lucigen makes some insulated vectors, and all of the Biobrick vectors have this isolation.
The vector is a lentiviral vector with a SFFV promoter, generally used for hematopoietic cells. Is this promoter active in E. coli??? Maybe a "residual" activity could cause this problem?
Thanks for the answer!
Posted 30 September 2010 - 03:16 PM