NO Insert found in the Colony PCR - (Jan/03/2012 )

You are right about the first one, i also know that, i don´t even get a good PCR product in it. I am not worried about the first one, but the others. They are quite ok as per the NEB standards. At least i should get a proper clone in them!!

Actually NcoI cutting site is - CCA TGG, so the NcoI will cut after the first C, its like C.....CA TGG. The first C is seperated. So there is a single nucleotidem you can not say that there is no nuclotode! But as per the litrature, minimum 3 nucleoties are should be present, but sometimes even 1 works.

I think i will take a proper look at the primers once again.... But thanks a lot!

The count is from the recognition site, not from the cut site. You have zero. The only primer that has extra bases is the NdeI site, with two. Even it won't cut very well, 0-20%, and the other end of that pcr product will not cut (EcoRI).

Well Veteran, thanks again for the reply. My situation is a little bad in this case, because i tried to explain this situation to the post doc in my laboratory who designed these primers, but she told me that she had designed similar primers before and they had worked and she told me that i should question my own work , so i guess i will continue ,my work and see what happens!

How much experience do you have in this kind of stuff?