I'm trying to linearize large amounts of plasmid (500 µg - 1000 µg). I can easily digest this amount of plasmid, but when I precipitate with ethanol and resuspend in TE, I'm left with a milky white solution. Initially I thought this was due to excess salt precipitation, but I can wash with 70% ethanol until the cows come home and it doesn't get any better, so clearly it's not salt.
I can purify with a column, but the losses are unacceptably high.
After butting my head against the wall for several days, I realized that my problem must be BSA from the restriction digest buffer. If I digest 1000 µg plasmid DNA in NEB cutsmart buffer, there's 5 mg BSA in the reaction. How can I remove the BSA so I'm not left with filthy dirty DNA when I precipitate? Will precipitating the BSA with ammonium acetate work?
You could probably clean it up by performing a Phenol-Chloroform extraction, followed by alcohol precipitation, if that would leave the plasmid suitable for your uses.
You mentioned that the losses in a column purification were too high, which seems odd. With some fiddling and optimization most plasmid purification columns can achieve around 90% recovery. Likewise, purification over a size-exclusion column can be pretty efficient - I had a good protocol using CL-4B Sepharose to separate most BSA from a plasmid sample.
I think my losses are from repeated alcohol precipitations, not from the efficiency of the column. The restriction digestion of 1000 µg of plasmid requires 50 ml total volume. Precipitating the plasmid from this brings the volume up to a little over 200 ml. This requires 8 tubes on my centrifuge. I lose a bit to each tube, leaving me at 60% to 70%.
I'm using anion exchange columns, because that's what I have on hand (maxiprep columns). The first time I did this I only had about 20% of my DNA left at the end after I eluted and precipitated to concentrate.
My thought process is that since I'm starting out with clean DNA, if I can just get rid the BSA then I only need to do the first precipitation. I'm fine with retaining 70%.
Will phenol-chloroform extraction work without first digesting the protein? It seems easier to just precipitate the protein. I've never done either, and I've already wasted more time than I want on this, so ...
In theory, a Phenol-Chloroform-Isoamyl-Alcholo wash step (PCIA) should remove the BSA without a previous proteinase K digestion. I'd say it's optional - especially if you don't already have Pro.K on hand.
Be aware that PCIA washing can cause some downstream issues because of phenolic contamination. I'm not sure what your final application is, so you might want to consider whether that will further complicate your work.
https://www.researchgate.net/post/How_does_one_remove_contaminants_from_phenolic_DNA_extracts
@ Bubacarr - I have 1000 µg of plasmid in 200 ml of water. That's a mighty big gel. Otherwise that's exactly what I would do.
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