I'm trying to purify a plasmid ~30kb in size, and the columns in the Qiagen mini-prep kit are giving pretty low (~1ng/uL) yields. Does anyone have a recommendation for another kit that is specific for plasmids 20 - 60 kb?
Hi I use the Machery-Nagel Nucleobond extra Plasmid midiprep kit to purify my plasmids of 35kb- these kits are designed specifically for larger plasmids. Hope this helps!
The Qiagen kit is probably your problem. I would definitely switch to a kit that is made for larger plasmids. While you are waiting for the new kit, you could try heating the elution buffer up to 70*C then add it to your column. Heat the column + buffer at 70*C for 5min, spin down at 50g for 1min then at 11,000g for 1min. Also rerun the elution buffer over your column at least 2 times because a lot of DNA remains on the column after the first elution
Try CIM DEAE column with 6 um pore size after the alkaline lysis to isolate pure plasmids. This is especially appropriate for plasmids above 15 kbp. And usually you get higher purities and higher yield compared to classical kits, because it is a chromatographic method of isolation and these columns have good chromatographic selectivity and resolution. Already CIM DEAE columns with 2 um pore size could give you excellent results, see the attached reference.
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