Make sure your homology arms are large enough? Have you tried different homology arm lengths?

try different bacteria? Try a different transformation method?

try PCRing the result of your Gibson to see if you even made the plasmid, because you don’t know if the issue is the assembly or the transformation.

Hello Sam Carter

What insert:vector ratios have you tried?

How much vector are you using? 18 KB is very large so you may need to scale up. A general guideline is 50 ng of plasmid for every 3 KB of size so you'd probably need at least 300 ng for each reaction.

Thank you Amy Klocko for your answer :). I've tried many different quantities of DNA from 100ng up to 300ng with ratio 1:1, 1:3, 1:10, 1:20.

Hi Julia Victoria Parsley the homology arms are 37bp long with the vector. My primers are 60bp in total. I tried 2 strains of bacteria but both with heat shock (Neb stable and XL10gold). I've read that electroporation is better for big plasmids but I'm not sure we have a bacteria strain that I can use for this. I definitely need to check if the problem is the gibson part or the transformation part, but I'm quite sure that it's the transformation because I have NEVER obtain any clone not even a false positive which never happened to me before. I always at least get a few colonies.

Hi Pushkal Sinduvadi Ramesh , I've tried many different ratios and quantities of DNA. Ratio 1:1, 3:1, 10:1 and 20:1 with vector quantity from 100ng to 300ng.

Sam Carter try electroporation definitely. Heat shock is finicky. Use DH10B strain E. Coli. thermo Fischer sells them

as a control, try cloning in the “empty” version of your plasmid with the heat shock. Then you can ensure the issue is the transformation. Always a good idea to pcr your Gibson result to confirm too. One primer binds in the insert one binds backbone. You only get the band if you made the plasmid.