Preferential insert orientation

bhjelle at unm.edu bhjelle at unm.edu
Thu Dec 30 11:53:41 EST 1993


I certainly have noticed this problem while "TA" cloning,
another seemingly symmetric cloning strategy. I wanted
clones in a particular orientation so I could use the
neighboring polylinker sites for subcloning. In one
example, 17 out of 17 clones went the wrong way!
Other inserts that shared the same 3' end (of the ORF)
refused to go in the correct orientation as well.

I suppose that small amounts of the protein product
of some ORFs might be made, and that that would be
toxic. In my case the insert was flanked by T7 and
SP6 promoters which should be quite silent in the
usual strains of coli. However, maybe lac Z or bla
was reading through.

The moral of the story is to use 2-enzyme ligations
whenever feasible. Or, consider subcloning into
some other vector with polylinker sites that 
make it easy to put the insert into the vector
you really want it in.

Good luck,
Brian  



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