alexis

I've chosen enzimes that produce sticky ends, and I used the same to cut the original vector and to amplify the promoter I want to clone, I don't know if it's clear the procedure I've followed. I don't know if my vector can religate, but the restriction site are different, so it's possible to religate even with different restriction sites? the problem is that I've got no colonies every times and I cannot verify the presence of the vector religated alone. because I've excised from this vector the original promotor, so if this vector is able to religate, without the promotot it cannot produce the Amp resistance, so I'vegot no colonies and I can't understand how to go on

Yes, I used the same enzimes to excise the original promotor from the bidirectional cassette, and to amplify the other promotor from its vector. I've used the same procedure to clone a 4.7 Kb insert into a 3.5 Kb vector and everything was OK, so this time I can't understand the problem using a smaller insert! Before starting the subsitution of the original promotor, I've checked the activity of the new promoter, and it was a not so strong promoter. I can't think that the new promoter is fatal, because the original promoter is a very strong one, it has been tested and there were no consecuences for the cells.

Hi everybody! I'm trying to substitute a 1 Kb promoter, from a bidirectional expression cassette, with another of 644 bp, from another vector. I've tried many times and I've done all the controls (efficiency of ligase, competent cells, restriction enzimes, ecc..): everything work, but every time I've got no colonies!! what could be the problem?? I'm at the first experiences with cloning and I'm desperate thanks to all! alexis