NEB Hifi DNA assembly kit is the best. Make sure to have atleast 30bases overlap between the insert and the vector.

NEB is the best way to go, but make sure follow the protocol...you can scale down their reaction size down to as low as 2.5 ul without impacting success rates or wasting a ton of reagents

Thanks @Rupsa Basu, @Amy Johnson, and @David Heisler. NEB it is! Unfortunately, ordered the original Gibson Kit (not HiFi). Is it worth trying with that or should I reorder?

Is glycogen pptn ok for (relatively nonabundant) PCR fragments for use in the assembly? Zymo gel isolation kit gives funky Nanospec readings (low 260/hi 230). Trying TE8 crush and soak w/glycogen ptn instead. (if no reason not to).

I cannot say I have ever done a glycogen soak. However, the high 230 is contamination from the buffer you use to dissolve the gel in. If the concentration of your fragment is high enough that you will be diluting it before putting in the actual reaction, I have found that its diluted out enough not to effect the HiFi reactions, can't say though for Gibson Kit.

Thanks David. Glycogen would be just as a carrier in precipitation. The issue at the moment is low concentration of product. Trying to increase PCR efficiency, but if can't will have to ppt. to concentrate (or I suppose use a kit and elute in a tiny volume (but eluting in about 10ul now with Zymo kit).

Also wondering:

Does DpnI work in Gibson reaction buffer?

Vector digestion appeared complete (so didn't gel purify pre Gibson), but apparently not. Wondering if futile to continue to screen (fragments limiting, so can't easily repeat...I've screened 20...control reaction worked), or if it would be possible to DpnI digest the reaction now to remove uncut vector, and retransform.

(I assume what I'm getting is uncut vector based on gel analysis...unless, there was some kind of recircularization and deletion).

DpnI will chew up all of your DNA minus whatever you produced by PCR. If you are only getting orginial plasmid then either a) you're digesting with only one enzyme that can be repaired by the Ecoli or b) more likely the digest is not complete and therefore the ecoli are keeping that plasmid over the Gibson combined as the original doesn't need repaired. I would suggest running uncut plasmid and the digestion on a low agarose gel and run it until you get a very clear separation between them and do the gel clean up

Thanks David Heisler for responding! Oh, of course, that's right, this wasn't a synthesis reaction, so can't use DpnI. Next shot, will definitely use gel purified vector. Vector was cut with NheI. My issue is unfortunately I can't immediately repeat (other fragments limiting). Is it worth continuing to screen colonies and transform the rest of this reaction? I realize it's not optimal, but might there be any assembled plasmid transformed at low frequency?

Sorry for the slow response. Yes I would have kept screening if the screening is fast/highthrough put enough.

Thanks!