My lab has the NEB Gibson Assembly kit (unfortunately not the Hifi kit) and I have been struggling for months to assemble various different constructs. I'm wondering if anyone has used this kit specifically and if there are any specific things/tricks you do or use?

I have tried to insert 3 fragments (ranging between 500 bp - 2 kb) into a vector backbone, as well as overlap PCRs to insert 1 fragment into the vector. All fragments have between 30-40 bp homology regions. I PCR amplify all of my pieces (including backbone) and gel extract them because we don't have DpnI. I can get the overlap PCR working well, so I thought the primers/homology regions were designed ok. Yet, I can't get a simple 1 insert into 1 vector reaction working - I consistently get zero colonies (occasionally I've gotten an absurdly low number like 2 or 4, but screening them they're false positives).

I've used NEB's positive control to make sure the Gibson mix is working and got many colonies, but unfortunately they don't provide much information on the contents other than it's 2 DNA fragments so I'm not sure how they designed the two fragments to be ligated.

I have typically been adding 50 ng vector and tried both equimolar amounts of insert and 2-3 fold molar excess of insert. Is there a concern that there is too much salt contamination carried over from the gel extraction? I was curious about the positive control NEB provides and nanodrop read quite comparable A260:A230 ratio between that and my fragment mix. I've also tried adding 5% DMSO into the reaction in case secondary structures were preventing efficient assembly, as well as transforming 50 uL of NEB10b with 2uL vs. 10uL of the Gibson reaction. And nothing seems to stick.

Sidenote I tried IVA once and didn't work for me, but if you have any specific recommendations with that I'd also be willing to try troubleshoot that method if I continue to have issues with Gibson.