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In this study, commercial anion exchange membrane and resin were used to purify plasmid DNA. After the removal of high molecular RNA in a pretreatment procedure, various alcohol concentrations in the washing buffer were examined for purification of plasmid DNA.
In batch experiments, we found that the increase of alcohol concentrations can enhance plasmid recovery and that the buffer containing 45% isopropanol had optimal purity and recovery.
When anion exchange membrane was used for purification of plasmid DNA, calcium was added in the pretreatment to remove high molecular weight RNA. The remaining RNA and protein can be removed by using a washing buffer containing 40% isopropanol and 1.5M NaCl. The purified plasmid DNA has a purity 100% and a recovery of 43±3%.
When anion exchange resin was used for purification of plasmid DNA, 1M guanidine-Cl and 50% isopropanol were added in the pretreatment to remove high molecular weight RNA. In small scale of purification process, a washing buffer containing 1M NaCl and 40% isopropanol was used. The obtained plasmid DNA has 100% purity and a recovery close to 100%. For plasmid purification by gravity-flow, the purification condition employed buffers containing 1.5M NaCl with step-down isopropanol concentrations. The plasmid DNA of different sizes obtained by this condition had 100% purity. When the gravity-flow method was used to purify 7.5 milliliter of fermentation broth, the recovery of 4.7kb and 8.0kb plasmid DNAs were 68.5±3% and 72±5%, respectively. When purification process was scaled up to 300 milliliter broth, the recovery of 4.7kb and 8.0kb plasmid DNAs were 75±6% and 50±3%, respectively. When purification process was scaled up to 3 liter broth, the recovery of 4.7kb and 8.0kb plasmid DNAs were 70% and 20%, respectively.
In conclusion, the use of commercial anion exchange membrane and resin coupled with washing buffer of various isopropanol concentrations can be used to obtain plasmid DNA of high purity and high recovery. Additionally, the use of regenerated anion exchange resin will not affect the purity of the purified plasmid DNA.
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