I have had trouble purifying very large quantities of pure PCR product. We are using these as templates for the reconstitution of nucleosomes and I require hundreds of micrograms to milligrams of template.
Here are some methods I have tried:
1) QiaexII slurry--typically I use the entire slurry for one purification then regenerate the resin by washing in 1M HCl then re-equilibrating with water. This way has worked best for me so far but the yield tends to be fairly low (~100 ug from 100 PCR reactions).
2) Phenol-chloroform extraction. This method seemed to work great, however I believe I have phenol contamination at the end--the yields are often much higher than they should be even if I chloroform back extract before the ethanol precipitation. When I run it out on a gel it looks like the apparent concentration I get is off. I have not tried this too many times so perhaps it could be optimized.
3) Spin column purification. I've used the DNA Clean and Concentrator from Zymo and the yields seemed quite low, even for the columns with an apparent 100 ug capacity.
Another option I have explored is large scale purification of the parent plasmid followed by digestion to liberate the small insert. However I still run into the issue of purifying away the parent plasmid. I've seen a few protocols using FPLC and/or size exclusion but I'm not sure how effective this would be.
Anyone have any ideas?