Hi :)

I'm currently trying to assemble a 4-part Gibson Cloning. I'm using the "NEBuilder Hifi DNA assembly master mix" for this. The backbone is 3kb long, the other fragments are 3,5kb, 3,6kb and 5kb long. First I designed the primers with this sofware on the NEB website, but the assembly didn't work. After reading some troubleshooting, I tried to design the overhangs with a Tm around 50°C. Since there is a NotI restriction site in one of the overhangs, the GC content is very high and the overhangs are now very short... It hasn't worked so far, there are no colonies after the transformation (controls work, the competent cells are ok). On an Agarose gel I can see that the fragments have not assembled... Has someone a good advice for me?

We thought of using a temperature gradient for the assembly (e.g. from 45°C to 55°C), but I don't know if this will change something...

Thanks :)