TOPO cloning always gets wrong orientation - (Sep/02/2006 )

I was hoping someone could help with my TOPO cloning which is supposed to be nondirectional (50% each orientation) but after three attempts and 25 colonies my insert is always in the reverse orientation. I've sent one in for sequencing and it is the correct insert, just backwards. Additionally, this insert is huge, 6kb insert into 4kb vector, so there's no restriction sites I can use to turn the insert around.. they all cut the insert. Any ideas?

If you are absolutely certain that there are no restriction sites that can be use in this instance to orientate the insert ....

Well I revert to the my lab's philosophy of 'Brute force and ignorance'

Test more colonies. Do colony PCR, with a multichannel pippette on a 96 well plate. Test 200 colonies if needed. It is even possible to screen 144 minipreps by hand in a day. So Brute force and Ignorance!

Alternatively.... have you checked your rare cutters like NotI, AscI, PacI or the hideously expensive FseI or even XmaI. Look up the rare cutter of NEB and see if any of these restriction enzymes can be used.

The orientation problem is due to tertiary structure which the insert has adopted. The only thing that can be done in this situation is to alter the ligation buffer salt concentration and risking failure and reduced efficiency of the ligation reaction.

Good luck. I have experience a similar orientation phenomena, except in my case the orientation was 90% in the correct direction.

I don't recall whether there's a promoter on the TOPO vectors, but could your gene be lethal to E. coli? If it were lethal, you'd only get opposite oriented clones, because the other clones would express the gene and kill the host.

I'm with HomeBrew. It sounds as if either transcription in or transcription out of our insert is the problem, and you are selecting against the orientation you want. Lucigen has a series of vectors with transcriptional terminators surrounding the cloning site, and we have made our own, for exactly this reason.