I'm currently trying to tag my gene of interest in-situ with a fluorescent tag and puro resistance gene replacing the stop codon using a TALEN based homologous recombination/repair approach.
My initial donor plasmid has some issues (due to the low expression of my gene of interest I don't think pyro is expressed in high enough quantities to give resistance), so I'm using Gibson Assembly to try to change the donor plasmid and include a promoter in front of my puro resistance and swap out the GFP tag for tdTomato.
I'm linearizing my backbone by PCR, and the Gibson reaction should be adding two fragments (both between 1-1.5kb). I've stepped the reaction time up to 60 min (from the 15 suggested in the manual) and am using equimolar concentrations of all fragments and vector.
My problem is, I get very few colonies when I transform our in house DH5alphas with a 1:4 dilution of the Gibson reaction (as suggested on the NEB instructions). Of those colonies I do get, none have the correct sequence. I've screened at least 30 colonies now to no avail. I should also mention, that I'm having very low numbers of resulting colonies from the positive control supplied with the NEB Gibson Mastermix.
Has anyone else had similar issues when using the Gibson method? Any help or advice would be greatly appreciated.
All the best,