I've been having a lot of trouble with gel purification in my lab, to such a degree that I don't think I've obtained a usable product from gel purification for at least a couple months. I've used NEB, Zymo, Sigma, and Machery-Nagel kits, and none have given satisfactory results. The Zymo and Machery-Nagel kits simply failed to recover any detectable DNA, while the Sigma kit recovers very little DNA, and a lot of 230 absorbers (have you ever seen a 260/230 ratio of 0.02? I have!).
The NEB Monarch kit recovers DNA at a low, but certainly usable concentration (~10-30 ng/ul) that is pretty dirty, but not stupendously dirty (260/230 ~ 1-1.5). However, when I try to use that DNA for anything - reamplification, for instance - it's unusable. Enzyme reactions fail, PCR returns a smear on gel, Gibson's don't assemble - either the DNA is damaged, or is contaminated with something that just ruins everything downstream, I don't know which. I use other Monarch kits for minipreps and reaction cleanup, and they work fine - no complaints at all. I always preheat the elution buffer, and I let it sit on the column for a few minutes before spinning, and I always do a dry spin after washes. I've also tried different gel percentages, I've tried different gel, including low-melt - doesn't make a lick of difference.
Now, I used to do gel purification all the time, some years ago. It was a standard part of my workflow, and I remember using both Qiagen and Sigma kits, and not paying too much attention to which one, because they always seemed to work to my satisfaction - clean DNA at concentrations of 100-300 ng/ul. But I was generally purifying fragments of 500-1500bp. I'm currently trying to purify fragments in the range 5-10kb. My colleague has said that he is convinced that all gel purification kits suck in general, but especially for high MW fragments, which he says is basically hopeless. But I remember gel purification being easy - so easy I would just load my entire PCR onto a gel directly from the thermocycler, confident that I would get pretty much all of my material back.
So do all gel purification kits suck in this size range? And what could I be doing differently to get usable product from gel purification? I'm especially curious about the results from the Monarch kit - judging by spec alone, it seems like it gets the job done, albeit with mediocre yield, but then anything I try to do downstream fails - why?
In case anyone asks, no, I'm not using UV to visualize, I'm using blue light.