Purifying large amounts of PCR product

Question

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?

Answer 1

We had to do this frequently. What I would first recognize is that all of your current methods only work for at the small scale and that for you to purify mg quantities of DNA, you need to work with mg scale equipment.

First of all, you should examine your "upstream" process. Are you doing your PCR at scale? Typically, we have scaled out by going with multiple PCR reactions rather than one large PCR reaction. Generally the reactions max out at 50uL depending on your thermocycler. If you're getting around 1ug/10uL, scale up to a 96 well plate with 50 uL reactions and get ~500 ug of material. Make sure that you mix your dNTPs! Scale-up accordingly.

To start, the DNA Clean and Concentration is designed for really small reactions. It's much better to use the next level of purification by maxiprepping everything. The The binding capacity of those columns go up to 1 mg. Using the QIAEX II slurry works but you should make sure that you scale up correctly. Make sure that you complete your regeneration. This protocol works well.

Alternatively you can ignore the column and just ethanol precipitate everything to remove your nucleotides. I suggest following Ethanol precipitation of nucleic acids from OWW. We had to make a lot of linear template and for most applications this works great.

As with all things related to molecular biology, there is a lot of voodoo. Some things that supposively work is to chill your ethanol mixture at -20C for at least an hour (or overnight), generally, you'll see your DNA fall out of solution and that's your sign that things are working. At this scale you should be seeing your DNA pellet; that's a good sign if things are working. While eluting anything from the columns, we would warm up our elution buffers. Thermodynamically this would make sense since it would reduce the barrier for unbinding. You would also get away with a lower residence time.

I would frankly avoid using FPLC unless you're working with gram quantities of DNA. You're not Moderna.