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- Syren, Per-Olof, et al.
(author)
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Milligram scale parallel purification of plasmid DNA using anion-exchange membrane capsules and a multi-channel peristaltic pump
- 2007
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In: Journal of chromatography. B. - : Elsevier B.V.. - 1570-0232 .- 1873-376X. ; 856, s. 68-74
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Journal article (peer-reviewed)abstract
- A parallel chromatog. procedure for the purifn. of milligram amts. of plasmid DNA was developed. Initial studies showed that ion-exchange membrane capsules displayed high capacity for plasmid DNA. Interestingly, a weak anion exchanger (DEAE) proved to be superior to the strong quaternary ammonium group with respect to elution and regeneration properties and the 75 cm2 Sartobind D membrane capsule (MA75D, Sartorius) was selected for further studies. A method for reducing endotoxin levels by using CTAB as a precipitant was optimized. By introducing this step into the protocol, endotoxin levels could be reduced approx. 100-fold to ≤5 EU/mg plasmid. The parallel procedure was set up on a multi-channel peristaltic pump and evaluated with four different vectors (2.7-11.5 kbp). Starting with 5-10 g of E. coli cell paste (wet wt.) generally satd. the membrane adsorber, resulting in plasmid DNA yields close to 10 mg. [on SciFinder(R)]
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